Practical applications of time-averaged restrained molecular dynamics to ligand-receptor systems: FK506 bound to the Q50R,A95H,K98I triple mutant of FKBP-13

Summary The ability of time-averaged restrained molecular dynamics (TARMD) to escape local low-energy conformations and explore conformational space is compared with conventional simulated-annealing methods. Practical suggestions are offered for performing TARMD calculations with ligand-receptor systems, and are illustrated for the complex of the immunosuppressant FK506 bound to Q50R,A95H,K98I triple mutant FKBP-13. The structure of ¹³C-labeled FK506 bound to triple-mutant FKBP-13 was determined using a set of 87 NOE distance restraints derived from HSQC-NOESY experiments. TARMD was found to be superior to conventional simulated-annealing methods, and produced structures that were conformationally similar to FK506 bound to wild-type FKBP-12. The individual and combined effects of varying the NOE restraint force constant, using an explicit model for the protein binding pocket, and starting the calculations from different ligand conformations were explored in detail.Abbreviations DG distance geometry - dmFKBP-12 double-mutant (R42K,H87V) FKBP-12 - FKBP-12 FK506-binding protein (12 kDa) - FKBP-13 FK506-binding protein (13 kDa) - HSQC heteronuclear single-quantum coherence - K_NOE force constant (penalty) for NOE-derived distance restraints - MD molecular dynamics - NOE nuclear Overhauser effect - SA simulated annealing - TARMD molecular dynamics with time-averaged restraints - tmFKBP-13 triple-mutant (Q50R,A95H,K98I) FKBP-13 - wtFKBP-12 wild-type FKBP-12     

Evaluation of coupling conditions for the incorporation of Nα-Fmoc-Tyr(PO3H2)-OH in solid-phase peptide synthesis

Summary In order to minimise the formation of the pyrophosphate derivative of the target peptide when side-chain-unprotected phopshotyrosine is used in solid-phase peptide synthesis, this building block can be incorporated using benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate/1-hydroxybenzotriazole/N-methylmorpholine (1:1:2.3) in the presence of a chaotropic salt (0.4 M LiCl in N-methyl-2-pyrrolidinone).Abbreviations BOP benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate - DIEA diisopropylethylamine - Fmoc 9-fluorenylmethoxycarbony - HATU N-[(dimethylamino)1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethan-aminium hexafluorophosphate N-oxide - HOBt 1-hydroxybenzotriazole - HPLC high-performance liquid chromatography - MALDI-TOF matrix-assisted laser-desorption ionization time-of-flight mass spectrometry - NMM N-methylmorpholine - NMP N-methyl-2-pyrrolidinone - Pmc 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl - ® solid support - TFA trifluoroacetic acid - TPTU 2-(2-pyridon-1-yl)-1,1,3,3-tetramethyluroniumfluoroborate. Abbreviations used for amino acids follow the recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9]     

A new mite-plant association: mites living amidst the adhesive traps of a carnivorous plant

Non-antagonistic interactions between arthropods and leaves of insectivorous plants with adhesive traps so far have never been reported. The mites are common prey of such plants, but we have found a new subspecies of the mite Oribatula tibialis living on the leaves of Pinguicula longifolia. Because of its small size and the low glandular density of the host, the mite moves without being trapped by the mucilaginous droplets of the leaf surface. P. longifolia provides shelter and food for the mite, while the plant may also benefit because of its fungivorous and scavenging activities. This new interaction is another dramatic example of widespread miteplant associations.     

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.     

Factors involved in capillary growth in the heart

Growth of capillaries in the heart occurs under physiological circumstances during endurance exercise training, exposure to high altitude and/or cold, and changes in cardiac metabolism or heart rate elicited by modification of thyroid hormone levels. Capillary growth in all these conditions can be linked with increased coronary blood flow, decreased heart rate, or both. This paper brings evidence that, although increased blood flow due to long-term administration of coronary vasodilators results in capillary growth, a long-term decrease in heart rate induced by electrical bradycardial pacing in rabbits and pigs, or by chronic administration of a bradycardic drug, alinidine, in rats, stimulates capillary growth with little or no change in coronary blood flow. Decreased heart rate results in increased capillary wall tension, increased end-diastolic volume and increased force of contraction, and thus stretch of the capillary wall. This could lead to release of various growth factors possibly stored in the capillary basement membrane. Correlation was found between capillary density (CD) and the levels of low molecular endothelial cell stimulating angiogenic factor (ESAF) both in rabbit and pig hearts with CD increased by pacing. There was no relation between expression of mRNA for basic fibroblast growth factor and CD in sham-operated and paced rabbit hearts. In contrast, mRNA for TGFß was increased in paced hearts, and the possible role of this factor in the regulation of capillary growth induced by bradycardia is discussed.     

Effects of water level fluctuations on phytoplankton in a river-floodplain lake system (Paraná River,Argentina)

Several aspects of community organization wereanalyzed comparatively in a small side-arm of theParaná River (Correntoso) and a shallowfloodplain lake (El Tigre) (31° 41 S and60° 42 W), in relation to the hydrology of thesystem. Taxonomic and morphological composition inthe river differed from that in the lake: the riverhad lower species richness (151 vs 218),different contributions of some Classes to totalspecies number (higher Cyano-, Zygo- andDiatomophyceae vs higher Chlorophyceae), anddiffent proportions of nannoplanktonic algae (67.5%vs 80.7%) and netplanktonic filamentousspecies (18.2% vs 4.2%). Phytoplanktonbiomass, higher in the lake than in the river due tothe retention time, was mostly dominated bynannoplankton and netplankton. Loticphytoplankton was dominated by typical fluvialspecies of Diatomophyceae (R-strategists). Riverconditions seem to maintain a subclimacticcommunity, which was little impacted by the flushingof populations from floodplain lakes. Water levelwas the main factor controlling phytoplanktonbiomass, species diversity (H), evenness (E) andcommunity change rate () in the river. Inthe lake, phytoplankton had an autogenicsuccessional sequence during the isolation phase (C-to S-strategists) and other responses todisturbance, mainly during the flood(R-strategists). Frequent changes in phytoplanktoncomposition, biomass, H, E and , revealed aenvironmental instability in the lake, which may beexplained by interactions of external factors(hydrology and climatology) and those of internalorigin, such as nutrients and grazing.     

TNF-alpha in smoke inhalation lung injury

Hales, Charles A., T. H. Elsasser, Peter Ocampo, and OlgaEfimova. TNF- in smoke inhalation lung injury.J. Appl. Physiol. 82(5):1433-1437, 1997.Adult respiratory distress syndrome is a majorcause of morbidity in fire victims. Tumor necrosis factor- (TNF-)is edematogenic and has been associated with the etiology of otherforms of adult respiratory distress syndrome. In the sheep lymphfistula model, we measured TNF- after 48 (n = 7) or 128 (n = 3) breaths of cotton smoke andcompared this with sham controls (n = 5) or controls in which left atrial pressure was elevated to 20 mmHg(n = 5) to increase lymph flow in the absence of inflammation. Smoke induced a rise in lymph flow and pulmonary arterial pressure with either no fall in lymph-to-plasma protein ratio (128 breaths) or a modest fall in lymph-to-plasma proteinratio (48 breaths), consistent with a change in microvascular permeability as well as a rise in microvascular pressure.Lymph concentration of TNF- fell in both groups, although lymph flux (concentration × flow) transiently rose in both. In neither case did TNF- flux exceed that induced by left atrial pressure elevation. TNF- was detectable in only one out of five sheep in alveolar lavage. Thus, by utilizing a sensitive and specific radioimmunoassay, we were unable to demonstrate a role for TNF- in smoke-induced microvascular lung injury in sheep.

     

Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers

Somaclonal variants resistant to sugarcane mosaic virus (SCMV) were obtained from susceptible sugarcane cv PR62258 through somatic embryogenesis by increasing the number of subcultures of the embryogenic callus tissue in MS medium with 3 mg/L 2,4-dichlorophenoxyacetic acid. Transfers were made at 30-day intervals for 1, 2 or 3 subcultures. Two somaclones, namely AT626 and BT627, were selected by their resistance to SCMV. These subclones have maintained the resistance trait over seven years of testing in the field. In this report we identified the somaclonal SCMV resistant variants from the maternal line and the nonresistant somaclones, using the RAPD technique.     

The larval development of lateral musculature in gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax

Fibre-type differentiation of the lateral musculature has been studied in Sparus aurata (L.) and Dicentrarchus labrax (L.) during larval development. Histochemical and ultrastructural techniques show two presumptive muscle layers and two germinative zones of presumptive myoblasts. At hatching, myotomal muscle consists of a monolayer of thin undifferentiated cells near the skin (first germinative zone) overlying another mono-layer of small diameter fibres extending hypaxially and epaxially away from the transverse septum. Below this, there is a much thicker, deep layer of fibres, generally large in diameter and polygonal in shape. The presumptive myoblasts are located between these two layers of fibres in the second germinative zone. Initially, the superficial and deep muscle fibres show high and low myosin ATPase activity, respectively. Both layers grow by generating new fibres from the two mentioned germinative zones. At the end of larval life, the superficial layer changes its histochemical profile from high to low myosin ATPase activity and, at the same time, intermediate or pink muscle fibres can be observed by oxidative activity (the NADH-TR reaction). Morphometric analysis shows a significant increase in mean fibre diameter during successive ages, as shown by the Student's t-test (hypertrophic growth). Skewness and kurtosis values of fibre diameters point to the generation of a new fibre population from the germinative zones (hyperplastic growth).