Unraveling the mechanism of the farnesyltransferase enzyme

Farnesyltransferase enzyme (FTase) is currently one of the most fascinating targets in cancer research. Studies in other areas, namely in the fight against parasites and viruses, have also led to very promising results. However, in spite of the thrilling achievements in the development of farnesyltransferase inhibitors (FTIs) over the past few years, the farnesylation mechanism remains, to some degree, a mystery. This review tries to shed some light on this puzzling enzyme by analyzing seven key mechanistic dilemmas, based on recent studies that have dramatically changed the way this enzyme is currently perceived.     

Distribution, structural and ecological aspects of the unusual leaf nectaries of Calolisianthus species (Gentianaceae)

Nectaries in leaves of Gentianaceae have been poorly studied. The present study aims to describe the distribution, anatomy, and ecological aspects of extrafloral nectaries (EFNs) of three Calolisianthus species and in particular the ultrastructure of EFNs in Calolisianthus speciosus during leaf development, discussing its unusual structure. Leaves of Calolisianthus species were fixed and processed by the usual methods for studies using light, scanning microscopy and transmission electron microscopy (TEM). Ion chromatography was used to analyze the nectar exudates of C. speciosus. The distribution patterns of nectar secretion units were analysed by ANOVA and t-tests. Two EFNs that can be seen macroscopically were observed at the bases of C. speciosus and C. pendulus leaves. Such large nectaries are absent there in C. amplissimus. Another similarly large EFN is observed at the apex of each leaf in all species. The EFNs at the base of the young leaves in C. speciosus are visited by ants during the rainy season. EFNs are formed by several nectar secretory units (nectarioles) that are present throughout the leaves. Each nectariole is formed by rosette cells with a central channel from which the nectar is released. Channels of old C. speciosus and C. pendulus EFNs were obstructed by fungi. TEM of EFNs in young leaves showed cytoplasms with secretion, small vacuoles, mitochondria, cell wall ingrowth, and plasmodesmata. TEM of EFNs in old leaves demonstrated dictyosomes, plastids, mitochondria, segments of endoplasmatic reticulum, and lipid droplets. The nectar contains sucrose, glucose and fructose.     

Unraveling the catalytic mechanism of nitrile hydratases

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k(cat) and K(m) for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK(E)(S1) and pK(E)(S2) values of 5.9 +/- 0.1 and 9.2 +/- 0.1 (k(cat)' = 130 +/- 1 s(-1)), respectively, and pK(E)(1) and pK(E)(2) values of 5.8 +/- 0.1 and 9.1 +/- 0.1 (k(cat)'/K(m)' = (6.5 +/- 0.1) x 10(3) s(-1) mm(-1)), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 degrees C, an Arrhenius plot was constructed by plotting ln(k(cat)) versus 1/T, providing E(a) = 23.0 +/- 1.2 kJ/mol. The thermal stability of PtNHase also allowed DeltaH(0) ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK(E)(S1) and pK(E)(S2) values. Based on DeltaH(0)(ion) data, pK(E)(S1) is assigned to betaTyr(68), whereas pK(E)(S2) is assigned to betaArg(52), betaArg(157), or alphaSer(112) (NHases are alpha(2)beta(2)-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.     

Unraveling the mechanism for respiratory rhythm generation

Breathing is generated by a neuronal network located within the caudal brainstem. One area of particular significance for respiratory rhythm generation is the pre-B?tzinger (preBotC) complex in the ventrolateral medulla. An important step towards understanding the cellular and network basis by which neurons within this region generate the respiratory rhythm was made in a recent study by Koshiya and Smith.(1) Using simultaneous image analysis and electrophysiological techniques these authors identified a discrete population of synaptically-coupled pacemaker neurons within the preBotC. They postulated that these neurons constitute the minimal essential network component (kernel) for generating the respiratory rhythm. BioEssays 22:6-9, 2000.     

Ultrastructure and post-floral secretion of the pericarpial nectaries of Erythrina speciosa (Fabaceae)

Background and Aims

The occurrence of nectaries in fruits is restricted to a minority of plant families and consistent reports of their occurrence are not found associated with Fabaceae, mainly showing cellular details. The present study aims to describe the anatomical organization and ultrastructure of the pericarpial nectaries (PNs) in Erythrina speciosa, a bird-pollinated species, discussing functional aspects of these unusual structures.

Methods

Samples of floral buds, ovaries of flowers at anthesis and fruits at several developmental stages were fixed and processed by the usual methods for studies using light, and scanning and transmission electron microscopy. Nectar samples collected by filter paper wicks were subjected to chemical analysis using thin-layer chromatography.

Key Results

The PNs are distributed in isolation on the exocarp. Each PN is represented by a single hyaline trichome that consists of a basal cell at epidermal level, stalk cell(s) and a small secretory multicellular head. The apical stalk cell shows inner periclinal and anticlinal walls impregnated by lipids and lignin and has dense cytoplasm with a prevalence of mitochondria and endoplasmic reticulum. The secretory cells show voluminous nuclei and dense cytoplasm, which predominantly has dictyosomes, rough endoplasmic reticulum, plastids, mitochondria and free ribosomes. At the secretory stage the periplasmic space is prominent and contains secretion residues. Tests for sugar indicate the presence of non-reducing sugars in the secretory cells. Nectar samples from PNs contained sucrose, glucose and fructose.

Conclusions

The secretory stage of these PNs extends until fruit maturation and evidence suggests that the energetic source of nectar production is based on pericarp photosynthesis. Patrolling ants were seen foraging on fruits during all stages of fruit development, which suggests that the PNs mediate a symbiotic relationship between ants and plant, similar to the common role of many extrafloral nectaries.     

Unraveling the catalytic mechanism of lactoperoxidase and myeloperoxidase.

Although belonging to the widely investigated peroxidase superfamily, lactoperoxidase (LPO) and myeloperoxidase (MPO) share structural and functional features that make them peculiar with respect to other enzymes of the same group. A survey of the available literature on their catalytic intermediates enabled us to ask some questions that remained unanswered. These questions concern controversial features of the LPO and MPO catalytic cycle, such as the existence of Compound I and Compound II isomers and the identification of their spectroscopic properties. After addressing each of these questions, we formulated a hypothesis that describes an integrated vision of the catalytic mechanism of both enzymes. The main points are: (a) a re-evaluation of the role of superoxide as a reductant in the catalytic cycle; (b) the existence of Cpd I isomers; (c) reciprocal interactions between catalytic intermediates and (d) a mechanistic explanation for catalase activity in both enzymes.     

Epigenetic memory in development and disease: Unraveling the mechanism

Epigenetic memory is an essential process of life that governs the inheritance of predestined functional characteristics of normal cells and the newly acquired properties of cells affected by cancer and other diseases from parental to progeny cells. Unraveling the molecular basis of epigenetic memory dictated by protein and RNA factors in conjunction with epigenetic marks that are erased and re-established during embryogenesis/development during the formation of somatic, stem and disease cells will have far reaching implications to our understanding of embryogenesis/development and various diseases including cancer. While there has been enormous progress made, there are still gaps in knowledge which includes, the identity of unique epigenetic memory factors (EMFs) and epigenome coding enzymes/co-factors/scaffolding proteins involved in the assembly of defined “epigenetic memorysomes” and the epigenome marks that constitute collections of gene specific epigenetic memories corresponding to specific cell types and physiological conditions. A better understanding of the molecular basis for epigenetic memory will play a central role in improving diagnostics and prognostics of disease states and aid the development of targeted therapeutics of complex diseases.     

The floral nectaries in theLimnanthaceae

Floral nectaries in theLimnanthaceae are established as exoscopic basal bulges of the episepalous stamens. Their nectariferous tissues include the epidermis and hypodermal parenchyma and inLimnanthes are vascularized by phloematic branches of the staminal bundles. Secretion occurs mainly through anomocytic stomata but, in addition, probably through the outer cuticularized thin walls of the epidermal cells. The flower structure is comparatively simple. The nectar is often slightly concealed. A wide range of pollinators can be expected, but bees are observed to be the dominant ones. The systematic position of the family is still obscure. Taxonomic placement near to any other geranialian families or to theCaryophyllaceae is only weakly justified.The floral nectaries of theGeraniales and their systematic implications II. For the first part seeLink (1992).     

Variability of the nectaries of the genusTurritis

Summary The monotype genusTurritis L. has lateral and median nectaries. The Iateral ones forming either a continuous ring or have the form of a horseshoe-shaped ridge open at the back. The median nectaries form a continuous ridge, sometimes enlarged in the median, sometimes being two-lobed.     

Unraveling the mechanism of radiosensitization by gemcitabine: the role of TP53

Gemcitabine has excellent radiosensitizing properties, as shown in both preclinical and clinical studies. Radiosensitization correlated with the early S-phase block of gemcitabine. In the present study, we investigated the role of TP53 in the radiosensitizing effect of gemcitabine. Isogenic A549 cells differing in TP53 status were treated with gemcitabine during the 24 h prior to irradiation. Cell survival was determined 7 days after irradiation by the sulforhodamine B test. In addition, cell cycle perturbation was determined by flow cytometry and TP53 expression by Western blot analysis. Gemcitabine caused a concentration-dependent radiosensitizing effect in all cell lines. Transformed A549 cells were less sensitive to the cytotoxic effect of gemcitabine. The cell cycle arrest early in the S phase was dependent on the drug dose but was comparable in the different cell lines and was not related to functional TP53. Using isogenic cell lines, we have shown that neither TP53 status nor the transfection procedure influenced the radiosensitizing effect of gemcitabine. Since both the radiosensitizing effect at equitoxic concentrations and the cell cycle effect of gemcitabine were independent of TP53 expression, it is likely that TP53 protein does not play a crucial role in the radiosensitizing mechanism of gemcitabine.     

Unraveling the mechanism of proton translocation in the extracellular half‐channel of bacteriorhodopsin

Bacteriorhodopsin, a light activated protein that creates a proton gradient in halobacteria, has long served as a simple model of proton pumps. Within bacteriorhodopsin, several key sites undergo protonation changes during the photocycle, moving protons from the higher pH cytoplasm to the lower pH extracellular side. The mechanism underlying the long‐range proton translocation between the central (the retinal Schiff base SB216, D85, and D212) and exit clusters (E194 and E204) remains elusive. To obtain a dynamic view of the key factors controlling proton translocation, a systematic study using molecular dynamics simulation was performed for eight bacteriorhodopsin models varying in retinal isomer and protonation states of the SB216, D85, D212, and E204. The side‐chain orientation of R82 is determined primarily by the protonation states of the residues in the EC. The side‐chain reorientation of R82 modulates the hydrogen‐bond network and consequently possible pathways of proton transfer. Quantum mechanical intrinsic reaction coordinate calculations of proton‐transfer in the methyl guanidinium‐hydronium‐hydroxide model system show that proton transfer via a guanidinium group requires an initial geometry permitting proton donation and acceptance by the same amine. In all the bacteriorhodopsin models, R82 can form proton wires with both the CC and the EC connected by the same amine. Alternatively, rare proton wires for proton transfer from the CC to the EC without involving R82 were found in an O′ state where the proton on D85 is transferred to D212. Proteins 2016; 84:639–654. © 2016 Wiley Periodicals, Inc.     

The floral nectaries in theIrvingiaceae

TheIrvingiaceae generally possess large intrastaminal receptacular disc nectaries of the mesenchymatic histo-type, which receive numerous small phloematic bundles directly from the central stele. The non-glandular epidermis bears some 10 to 15 strictly localized stomata that are deeply sunken in the parenchyma. The nectar is assumed to be exposed on the disc surface. Flowers are of simple construction, lacking specialized organs to attract pollinators. A wide range of pollinators is thus expected. TheIrvingiaceae have more characters in common withSimaroubaceae thanIxonanthaceae and should therefore be retransferred as a family of their own next toSimaroubaceae. The floral nectaries of theGeraniales and their systematic implications V. For the fourth part seeLink (1992)     

Unraveling the factors and mechanism involved in persistence: Host-pathogen interactions in Helicobacter pylori

Helicobacter pylori and humans have one of the most complex relationships in nature. How a bacterium manages to live in one of the harshest and hostile environments is a topic of unraveling mysteries. H. pylori is a prevalent species and it colonizes the human gut of more than 50% of the world population. It infects the epithelial region of antrum and persists there for a long period. Over the time of evolution, H. pylori has developed complex strategies to extend the degree of inflammation in gastric mucosa. H. pylori needs specific adaptations for initial colonization into the host environment like helical shape, flagellar movement, chemotaxis, and the production of urease enzyme that neutralizes acidic environment of the stomach. There are several factors from the bacterium as well as from the host that participate in these complex interactions. On the other hand, to establish the persistent infection, H. pylori escapes the immune system by mimicking the host antigens. This pathogen has the ability to dodge the immune system and then persist there in the form of host cell, which leads to immune tolerance. H. pylori has an ability to manipulate its own pathogen-associated molecular patterns, which leads to an inhibition in the binding with specific pattern recognition receptors of the host to avoid immune cell detection. Also, it manipulates the host metabolic homeostasis in the gastric epithelium. Besides, it has several genes, which may get involved in the acquisition of nutrition from the host to survive longer in the host. Due to the persistence of H. pylori, it causes chronic inflammation and raises the chances of gastric cancer. This review highlights the important elements, which are certainly responsible for the persistence of H. pylori in the human host.