Effect of the phosphate substrate on drug-inhibitor binding to human purine nucleoside phosphorylase

The thermodynamics of the drug-inhibitors acyclovir, ganciclovir, and 9-benzylguanine binding to human purine nucleoside phosphorylase (hsPNP) were determined from isothermal titration calorimetry as a function of the substrate phosphate ion (Pi) concentration from 0 to 0.125 M and temperature from 15 °C to 35 °C. At 25 °C and with an increase in the Pi concentration from 0 to 50 mM, acyclovir binding becomes more entropically-driven and ganciclovir binding becomes more enthalpically-driven. At 25 °C, the tighter 9-benzylguanine binding reaction goes from an enthalpically-driven reaction in the absence of Pi to an entropically-driven reaction at 10 mM Pi, and the enthalpically-driven nature of the binding reaction is restored at 75 mM Pi. Since the dependencies of the driving-nature of the binding reactions on Pi concentration can be simulated by Pi binding to its catalytic site, it is believed that bound Pi affects the interactions of the side-chains with the ribose catalytic site. However, the binding constants are unaffected by change in the bound Pi concentration because of enthalpy-entropy compensation. The enzymatic activity of hsPNP was determined by an ITC-based assay employing 7-methylguanosine and Pi as the substrates. The heat of reaction determined from the assay increased by 7.5 kJ mol⁻¹ with increase in Pi concentration from 50 to 100 mM and is attributed to weak binding of the Pi to a secondary regulatory site. Although the binding constants of acyclovir and ganciclovir at 20 μM hsPNP were in agreement with the inverse inhibition constants determined from the ITC enzyme inhibition assays at 60 nM, the binding constant of 9-benzylguanine, which interacts with Phe159 from an adjacent subunit, decreased from 5.62 × 10⁵ M⁻¹ to 1.14 × 10⁵ M⁻¹. This reduction in the 9-benzylguanine binding affinity along with a 7-fold increase in the specific activity of hsPNP at 14.5 nM results from partial dissociation of the hsPNP trimer into monomers below the 60 nM level.     

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-α-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.     

Changes in photosynthetic capacity and polypeptide patterns during natural senescence and rejuvenation of <Emphasis Type="Italic">Cucurbita pepo</Emphasis> L. (zucchini) cotyledons

Natural senescence of Cucurbita pepo (zucchini) cotyledons was accompanied by a gradual degradation of reserve proteins (globulins) and an intensive decrease in the content of both large subunit (LSU) and small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The net photosynthetic rate, the primary photochemical activity of PSII, estimated by the variable fluorescence (F_v)/maximal fluorescence (F_m) ratio (F_v/F_m) and the actual quantum yield of PSII electron transport in the light-adapted state (ΦPSII) also progressively decreased during natural senescence. In contrast, the fraction of the absorbed light energy, which is not used for photochemistry (LNU) increased with progression of senescence. The decline in the photosynthetic rate started earlier in ontogenesis compared with the down-regulation of the functional activity of PSII, thus suggesting the existence of protective mechanisms which maintain higher efficiency of the photochemical electron transport reactions of photosynthesis compared with the dark reactions of the Calvin cycle during earlier stages of natural senescence. Decapitation of the epicotyl above the senescing cotyledons resulted in full recovery of the polypeptide profile in the rejuvenated cotyledons. In addition, the photosynthetic rate increased reaching values that exceeded those measured in juvenile cotyledons. The photochemical efficiency of PSII also gradually recovered, although it did not reach the maximum values measured in the presenescent cotyledons.     

\Cytokinin oxidase/dehydrogenase (CKX) activity in wild and ethylene-insensitive mutant eti5 type of Arabidopsis thaliana (L.) Heynh plants and the effect of cytokinin N1-(2-chloro-4-pyridyl)-N2-phenylurea on enzymatic activity and leaf morphology

The specific activity of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) (CKX) was determined in leaves of wild type (wt) and ethylene-insensitive mutant (eti5) of Arabidopsis thaliana (L.) Heynh plants. Comparative studies showed that this mutation has lower basal CKX activity than wt. Application of 4PU-30 (N¹-(2-chloro-4-pyridyl)-N²-phenylurea) resulted in decreased CKX activity in both wt and mutant plants. The treatment increased leaf blade thickness and the volume of chlorophyll-containing cells per unit leaf area in wt but these changes were not observed in the eti5 mutant. The reduction in chlorophyll “a” and “b”, as well as in carotenoids content in the treated wt tissues resulting from altered leaf morphology was not detected in eti5 plants.     

The Maize Oil Yellow1 (Oy1) Gene Encodes the I Subunit of Magnesium Chelatase

Semi-dominant Oil yellow1 (Oy1) mutants of maize (Zea mays) are deficient in the conversion of protoporphyrin IX to magnesium protoporphyrin IX, the first committed step of chlorophyll biosynthesis. Using a candidate gene approach, a cDNA clone was isolated that was predicted to encode the I subunit of magnesium chelatase (ZmCHLI) and mapped to the same genetic interval as Oy1. Allelic variation was identified at ZmCHLI between wild-type plants and plants carrying semi-dominant alleles of Oy1. These differences revealed putative amino acid substitutions that could account for the alterations in protein function. Candidate lesions were tested by introduction of homologous changes into the Synechocystis magnesium chelatase I gene (SschlI) and characterization of the activity of mutant protein variants in an in vitro enzyme activity assay. The results of these analyses suggest that SsChlI protein variants containing the substitutions identified in the dominant Oy1 maize alleles lack activity necessary for magnesium chelation and confer a semi-dominant phenotype via competitive inhibition of wild-type SsChlI.     

A putative cation channel, NCA-1, and a novel protein, UNC-80, transmit neuronal activity in C. elegans

Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the α1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons.     

NaCl induced cross-acclimation to UV-B radiation in four Barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars

The effect of pre-treatment with 200 mM NaCl on the response of four barley cultivars (Hordeum vulgare L. cv. Bülbül-89, Kalaycı-97, Tarm-92 and Tokak-157/37) to UV-B radiation was investigated. Salt stress as well as UV-B irradiation led to a decrease of the total chlorophyll (chl) content in all cultivars, except in Kalaycı-97. While carotenoids are almost not affected by NaCl treatment, UV-B irradiation caused an increase by 5–20% of carotenoid content of all cultivars. UV-B induced damages of photosynthetic apparatus were estimated by the rate of photosynthetic electron transport measured by chl fluorescence and the rate of oxygen evolution, the latter being more affected. Pre-treatment with NaCl alleviated harmful effect of UV-B irradiation on F _v/F _m and ETR, but not on oxygen evolution. UV-B-induced and UV-B-absorbing compounds with absorption at 300 and 438 nm increased as a result of UV-B treatment. The level of stress marker proline increased considerably as a result of NaCl treatment, while UV-B irradiation resulted in a pronounced increase of the level of H₂O₂. MDA enhanced in the seedlings subjected to salt and UV-B stress. Established cross-acclimation to UV-B as a result of salt treatment could be due to the increased free proline and the level of UV-B absorbing compounds in barley seedlings subjected to NaCl.     

Growth form evolution and hybridization in Senecio (Asteraceae) from the high equatorial Andes

Changes in growth forms frequently accompany plant adaptive radiations, including páramo–a high‐elevation treeless habitat type of the northern Andes. We tested whether diverse group of Senecio inhabiting montane forests and páramo represented such growth form changes. We also investigated the role of Andean geography and environment in structuring genetic variation of this group. We sampled 108 populations and 28 species of Senecio (focusing on species from former genera Lasiocephalus and Culcitium) and analyzed their genetic relationships and patterns of intraspecific variation using DNA fingerprinting (AFLPs) and nuclear DNA sequences (ITS). We partitioned genetic variation into environmental and geographical components. ITS‐based phylogeny supported monophyly of a Lasiocephalus‐Culcitium clade. A grade of herbaceous alpine Senecio species subtended the Lasiocephalus‐Culcitium clade suggesting a change from the herbaceous to the woody growth form. Both ITS sequences and the AFLPs separated a group composed of the majority of páramo subshrubs from other group(s) comprising both forest and páramo species of various growth forms. These morphologically variable group(s) further split into clades encompassing both the páramo subshrubs and forest lianas, indicating independent switches among the growth forms and habitats. The finest AFLP genetic structure corresponded to morphologically delimited species except in two independent cases in which patterns of genetic variation instead reflected geography. Several morphologically variable species were genetically admixed, which suggests possible hybrid origins. Latitude and longitude accounted for 5%–8% of genetic variation in each of three AFLP groups, while the proportion of variation attributed to environment varied between 8% and 31% among them. A change from the herbaceous to the woody growth form is suggested for species of high‐elevation Andean Senecio. Independent switches between habitats and growth forms likely occurred within the group. Hybridization likely played an important role in species diversification.     

Estimating the Accuracy of Protein Structures using Residual Dipolar Couplings

It has been commonly recognized that residual dipolar coupling data provide a measure of quality for protein structures. To quantify this observation, a database of 100 single-domain proteins has been compiled where each protein was represented by two independently solved structures. Backbone ¹H–¹⁵N dipolar couplings were simulated for the target structures and then fitted to the model structures. The fits were characterized by an R-factor which was corrected for the effects of non-uniform distribution of dipolar vectors on a unit sphere. The analyses show that favorable values virtually guarantee high accuracy of the model structure (where accuracy is defined as the backbone coordinate rms deviation). On the other hand, unfavorable values do not necessarily suggest low accuracy. Based on the simulated data, a simple empirical formula is proposed to estimate the accuracy of protein structures. The method is illustrated with a number of examples, including PDZ2 domain of human phosphatase hPTP1E. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.