Investigating the Effect of Chain Connectivity on the Folding of a Beta-Sheet Protein On and Off the Ribosome

Determining the relationship between protein folding pathways on and off the ribosome remains an important area of investigation in biology. Studies on isolated domains have shown that alteration of the separation of residues in a polypeptide chain, while maintaining their spatial contacts, may affect protein stability and folding pathway. Due to the vectorial emergence of the polypeptide chain from the ribosome, chain connectivity may have an important influence upon cotranslational folding. Using MATH, an all β-sandwich domain, we investigate whether the connectivity of residues and secondary structure elements is a key determinant of when cotranslational folding can occur on the ribosome. From Φ-value analysis, we show that the most structured region of the transition state for folding in MATH includes the N and C terminal strands, which are located adjacent to each other in the structure. However, arrest peptide force-profile assays show that wild-type MATH is able to fold cotranslationally, while some C-terminal residues remain sequestered in the ribosome, even when destabilized by 2–3?kcal?mol^?1. We show that, while this pattern of Φ-values is retained in two circular permutants in our studies of the isolated domains, one of these permutants can fold only when fully emerged from the ribosome. We propose that in the case of MATH, onset of cotranslational folding is determined by the ability to form a sufficiently stable folding nucleus involving both β-sheets, rather than by the location of the terminal strands in the ribosome tunnel.     

Occurrence and diversity of mesophilic Shewanella strains isolated from the North-West Pacific Ocean

Although bacteria of the genus Shewanella belong to one of the readily cultivable groups of "Gammaproteobacteria", little is known about the occurrence and abundance of these microorganisms in the marine ecosystem. Studies revealed that of 654 isolates obtained from marine invertebrates (ophiuroid Amphiopholis kochii, sipuncula Phascolosoma japonicum, and holothurian Apostichopus japonicus, Cucumaria japonica), seawater and sediments of the North-West Pacific Ocean (i.e. the Sea of Japan and Iturup Is, Kurile Islands), 10.7% belonged to the genus Shewanella. The proportion of viable Shewanella species varied from 4% to 20% depending on the source of isolation. From the isolation study, representative strains of different phenotypes (from seventy presumptive Shewanella strains) were selected for detailed characterization using phenotypic, chemotaxonomic, and phylogenetic testing. 16S rDNA sequence-based phylogenetic analysis confirmed the results of tentative identification and placed the majority of these strains within only a few species of the genus Shewanella with 98-99% of 16S rDNA sequences identity mainly with S. japonica and S. colwelliana, suggesting that the strains studied might belong to these species. Numerically dominant strains of S. japonica were metabolically active and produced proteinases (gelatinases, caseinases), lipases, amylases, agarases, and alginases. Shewanella strains studied demonstrated weak antimicrobial and antifungal activities that might be an indication of their passive role in the colonization on living and non-living surfaces.     

Body mass changes in waders (Charadrii) in a high arctic area at northern Taimyr, Siberia

Body mass data were collected for 5 species of tundra waders during 6 breeding seasons to study variation in nutrient reserves relative to high arctic climatic conditions and the stage of reproduction. Structural size was accounted for by the 1st Principal Component of external linear measurements: wing length, bill length and tarsus length. SandpipersCalidris spp. were on average lighter in phenologically late seasons, while mass of Turnstones (Arenaria interpres) was similar in all years. Mass of waders was lower during brood-rearing than during incubation. Body mass during pre-nesting was most variable between years, reflecting the unpredictable weather conditions in this period. Monogamous species (KnotsCalidris canutus and Turnstones) began to breed earlier, and were on average, as heavy during the pre-nesting period as during incubation. In contrast, species with a proven or suspected rapid double-clutch breeding system (Little StintsC. minuta and SanderlingsC. alba) began to breed later and had during the laying period masses close to those of adults attending broods, and lower than during incubation. Body mass of the two latter species and Curlew Sandpipers (Calidris ferruginea) varied during incubation, reaching peak at 7 to 13 days after clutch completion.

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Zusammenfassung Von fünf Tundra-Watvogelarten wurden in sechs Brutperioden Daten zur Körpermasse gewonnen, um die Dynamik der Energiereserven in Abhängigkeit von den klimatischen Bedingungen der Hocharktis und vom Fortpflanzungsstadium zu analysieren. Der Einfluß der Körpermaße auf die Masse wurde bei der statistischen Auswertung durch die Einbeziehung der 1. Hauptkomponente von Flügel-, Schnabel- und Tarsuslänge berücksichtigt. StrandläuferCalidris spec. erwiesen sich in späten Jahren als leichter, während in der Körpermasse von Steinwälzern (Arenaria interpres) keine Unterschiede festgestellt werden konnten. Die Körpermasse aller untersuchter Arten war während der Betreuung der Jungvögel geringer als während Brütezeit. Die Masse in der Zeit vor der Brut variierte in Übereinstimmung mit den wechselnden Wetterbedingungen in den einzelnen Jahren am stärksten. Monogame Arten (KnutCalidris canutus und Steinwälzer) begannen früher mit der Fortpflanzung; ihre Körpermasse in der Phase von der Brut und während der Brütezeit war im Mittel etwa gleich. Im Gegensatz dazu begannen Arten, bei denen ein Doppelbrutsystem nachgewiesen wurde oder vermutet wird (ZwergstrandläuferC. minuta und SanderlingC. alba), später mit dem Nestbau, wobei die Körpermasse auf ein Niveau absinkt, das dem während der Betreuung der Jungvögel nahekommt und geringer ist, als das während der Brütezeit. Bei den beiden letzten Arten sowie beim Sichelstrandläufer (Calidris ferrugunea) veränderte sich die Körpermasse während des Brütens und erreichte ein Maximum zwischen dem 7. und dem 13. Tag nach Beendigung der Eiablage.

     

Crystal structure of head-to-head dimers of cholic and deoxycholic acid derivatives with different symmetric bridges

The crystal structure of three head-to-head dimers (having two cholic acid or deoxycholic acid units) linked at carbon atoms C3 by aromatic or alkyl bridges is studied. An internal coordinates system is necessary for describing the relative orientation in the space of the two bile acid residues. Five angles (three torsion and two common ones) are necessary for defining the relative position of both steroid residues in space. Carbon atoms C3 (which always carries a α-hydroxy group in natural bile acids), and C10 and C13 (which always carry β-methyl groups) of each steroid residue are suitable for this purpose. Furthermore, the distance between each C3 carbon atoms of both steroid residues will allow one to locate the steroids in space. The three dimers selected provide a large range of values for these angles. The packing, hydrogen bond network, and location of guest in the three crystals are discussed.     

Separation and quantification of ropinirole and some impurities using capillary liquid chromatography

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinson’s disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary liquid chromatography (CLC) was used for the separation and quantification of ropinirole and its five related impurities, potentially formed during its synthesis. A simultaneous optimization of three mobile phase parameters, i.e., pH, buffer concentration and acetonitrile content was performed employing an experimental design approach which proved a powerful tool in method development. The retention factors of the investigated substances in different mobile phases were determined. Baseline resolution of the six substances on a C₁₈ reversed stationary phase was attained using a mobile phase with an optimized composition [acetonitrile–8.7 mM 2-(N-morpholino)ethanesulfonic acid adjusted to pH 6.0 (55:45, v/v)]. It was shown that CLC, operated in the isocratic mode under the mobile phase flow-rate of 4 μl/min, can determine the level of these impurities, down to a level of 0.06% of the main component within 25 min.     

Vasotocin, melatonin and narcolepsy: Possible involvement of the pineal gland in its patho-physiological mechanism

Both the pineal nonapeptide hormone arginine vasotocin (AVT) (2.5 μg) administered intra-nasally and the pineal indole melatonin (50 mg) administered intravenously to three male narcoleptics (two with auxiliary symptoms and one with sleep attacks only), dramatically increased the amount of REM sleep and decreased REM sleep latency. The duration of the sleep onset REM periods in the two narcoleptics with auxiliary symptoms increased by more than 100 percent after AVT and melatonin administration. In the narcoleptic with sleep attacks only both AVT and melatonin induced REM periods at sleep onset. The hypothesis is advanced that narcolepsy represents an impairment of the melatonin-AVT control in the induction and circadian organization of REM sleep associated with an immaturity of REM triggering centers.     

Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated Na i + /K i + -independent signaling

Recent studies demonstrate that cytotoxic actions of ouabain and other cardiotonic steroids (CTS) on renal epithelial cells (REC) are triggered by their interaction with the Na⁺,K⁺-ATPase α-subunit but not the result of inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio. This study examined the role of mitogen-activated protein kinases (MAPK) in the death of ouabain-treated REC. Exposure of C7-MDCK cells that resembled principal cells from canine kidney to 3 μM ouabain led to phosphorylation of p38 without significant impact on phosphorylation of ERK and JNK MAPK. Maximal increment of p38 phosphorylation was observed at 4 h followed by cell death at 12 h of ouabain addition. In contrast to ouabain, neither cell death nor p38 MAPK phosphorylation were affected by elevation of the [Na⁺]_i/[K⁺]_i ratio triggered by Na⁺,K⁺-ATPase inhibition in K⁺-free medium. p38 phosphorylation was noted in all other cell types exhibiting death in the presence of ouabain, such as intercalated cells from canine kidney and human colon rectal carcinoma cells. We did not observe any action of ouabain on p38 phosphorylation in ouabain-resistant smooth muscle cells from rat aorta and endothelial cells from human umbilical vein. Both p38 phosphorylation and death of ouabain-treated C7-MDCK cells were suppressed by p38 inhibitor SB 202190 but were resistant to its inactive analogue SB 202474. Our results demonstrate that death of CTS-treated REC is triggered by Na_i⁺,K_i⁺—independent activation of p38 MAPK.