Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.     

A novel imidazole nucleoside containing a diaminodihydro-S-triazine as a substituent: inhibitory activity against the West Nile virus NTPase/helicase

The attempted synthesis of a ring-expanded guanosine (1) containing the imidazo[4,5-e][1,3]diazepine ring system by condensation of 1-(2'-deoxy-beta-D-erythropentofuranosyl)-4-ethoxycarbonylimidazole-5-carbaldehyde (2) with guanidine resulted in the formation of an unexpected product, 1-(2'-deoxy-beta-D-erythropentofuranosyl)-5-(2, 4-diamino-3, 6-dihydro-1,3, 5-triazin-6-yl)imidazole-4-carboxamide (7). The structure as well as the pathway of formation of 7 was corroborated by isolation of the intermediate, followed by its conversion to the product. Nucleoside 7 showed promising in vitro anti-helicase activity against the West Nile virus NTPase/helicase with an IC50 of 3-10 microg/mL.     

Low Night Temperature-Induced Changes in Photosynthesis and Rubber Accumulation in Guayule (Parthenium Argentatum Gray)

Photosynthetica - Three-year-old plants of Parthenium argentatum Gray cv. 11591 grown under natural photoperiod were exposed for 60 d to low night temperature (LNT) of 15 °C (daily from 18:00...     

Options to avoid the second surgical site: a review of literature

As esthetics gain importance, periodontal plastic surgical procedures involving soft tissue grafts are becoming commoner both around natural teeth as well as around implants. Periodontal soft tissue grafts are primarily used for the purpose of root coverage and in pre-prosthetic surgery to thicken a gingival site or to improve the crestal volume. Soft tissue grafts are usually harvested from the palate. Periodontal plastic surgical procedures involving soft tissue grafts harvested from the palate have two surgical sites; a recipient site and another donor site. Many patients are apprehensive about the soft tissue graft procedures, especially the creation of the second/donor surgical site in the palate. In the past decade, newer techniques and products have emerged which provide an option for the periodontist/patient to avoid the second surgical site. MucoMatrixX, Alloderm^®, Platelet rich fibrin, Puros^® Dermis and Mucograft^® are the various options available to the practicing periodontist to avoid the second surgical site. Use of these soft tissue allografts in an apprehensive patient would decrease patient morbidity and increase patient’s acceptance towards periodontal plastic surgical procedures.     

Quantitative assessment of the mutagenic effect of ethyl methanesulfonate inMicrococcus glutamicus

Micrococcus glutamicus ATCC 13032, a glutamic acid-excreting organism, was mutated by treating a 6–8 h old culture with 0.2m ethyl methanesulfonate (EMS) in a phosphate buffer of pH 7.6 for 150 min. The auxotrophs isolated showed varied patterns of extracellular amino acids, 38 isolates excreted lysine up to 1–2 mg/ml but most lost their excretion potency in subsequent fermentation trials although they remained auxotrophic. Most of the auxotrophs showed a requirement for amino acids.     

Computational identification of putative miRNAs and their target genes in pathogenic amoeba Naegleria fowleri

Naegleria fowleri is a parasitic unicellular free living eukaryotic amoeba. The parasite spreads through contaminated water and causes primary amoebic meningoencephalitis (PAM). Therefore, it is of interest to understand its molecular pathogenesis. Hence, we analyzed the parasite genome for miRNAs (microRNAs) that are non-coding, single stranded RNA molecules. We identified 245 miRNAs using computational methods in N. fowleri, of which five miRNAs are conserved. The predicted miRNA targets were analyzed by using miRanda (software) and further studied the functions by subsequently annotating using AmiGo (a gene ontology web tool).     

Synthetic Approaches to 5:8-Fused Heterocyclic Systems. A Novel Rearrangement During the Synthesis of Imidazo[4,5-e][1,2,4]triazocine Ring System

Abstract

The attempted ring-closure of 4-amino-1-benzyl-5-[(N²-benzyl-N²-methoxy-carbonyl)hydrazinomethylenecarbonyl]imidazole (5b) in sodium hydride/dimethyl sulfoxide at 50–60 °C afforded 1,7-dibenzylxanthine (6) instead of the anticipated 5:8-fused heterocycle 4b. A tentative reaction pathway has been proposed for the observed transformation.     

Egg-derived tyrosine phosphatase as a Potential Biomarker for Muscle Ageing and Degeneration in Drosophila melanogaster

Ageing is associated with declined activity of behaviors, physiology and metabolic processes （Arking, 2006）. In- vestigations in model organisms have indicated the exis- tence of ＂functional senescence＂, the progressive decline of biological functions with age and the decline in the activity may vary from tissue to tissue. Consequently, studies per- taining to the key organs/tissues whose functions deterio- rate/fail with age have led to the development of tissue specific ageing biomarkers （Grotewiel et al., 2005; Demontis et al., 2013）.     

Diversity and ecology of algae from the Nahal Qishon river,northern Israel

In 59 samples of periphyton and phytoplankton collected in 2002 - 2003 from the Nahal Qishon (Qishon River), northern Israel, we found 178 species from seven divisions of algae and cyanoprocaryotes. Diatoms, clorophytes, and cyanoprocaryotes prevail. Nitzschia and Navicula (Bacillariophyta) are the most abundant. Most of the species are cosmopolitan or widespread, except Lagynion janei (Chrysophyta), which is endemic for the Mediterranean Realm. About 17% of species (26) are new for Israel and five of them represent the first recorded genera: Crinalium endophyticum Crow, Actinocyclus normanii (Gregory) Hustedt, Rhizoclonium hieroglyphicum (Agardh) Kütz (Chlorophyta), Lagynion janei Bourelly, and Stylococcus aureus Chodat. Most of them come from a rare riverine assemblage with red alga Audouinella pygmea, as well as from the estuarine assemblage. Alkaliphiles predominate among the indicators of acidity, with few acidophiles confined to the communities under the impact of industrial wastes. Among the indicators of salinity, most numerous are the oligohalobien-indifferents and species adapted to a moderate salinity level. The relative species richness of ecological groups and the indices of saprobity are correlated with changes in conductivity, pH, and N-nitrate concentration. Indicators of organic pollution fall in the range of betameso- to alfamesosaprobic self-purification grades. Our studies show ecological significance of the Nahal Qishon as a model for a strongly disturbed aquatic ecosystem in the coastal zone of eastern Mediterranean.