βVI Turns in peptides and proteins: A model peptide mimicry

To investigate the role of proline in defining β turn conformations within cyclic hexa- and pentapeptides we synthesized and determined the conformations of a series of L - and D -proline-containing peptides by means of 2D NMR spectroscopy and restrained molecular dynamics simulations. Due to cis/trans isomerism the L -proline peptides adopt at least two different conformations that are analyzed and compared to the structures of the corresponding D -proline peptides. The cis conformations of the compounds cyclo(-Pro-Ala-Ala-Pro-Ala-Ala-), cyclo(-Arg-Gly-Asp-Phe-Pro-Gly-), cyclo(-Arg-Gly-Asp-Phe-Pro-Ala-), cyclo(-Pro-Ala-Ala-Ala-Ala--), and cyclo(-Pro-Ala-Pro-Ala-Ala-) form uncommon βVI turns that mimic the turn geometries found in crystallographically refined protein structures at such a detailed level that even preferred side chain orientations are reproduced. The ratios of the cis/trans isomers are analyzed in terms of the steric demand of the proline-following residue. The conformational details derived from this study illustrate the importance of the examination of small model compounds derived from protein loop regions, especially if bioactive recognition sequences, such as RGD (Arg-Gly-Asp), are incorporated. © 1993 Wiley-Liss, Inc.     

Species composition,abundance and habitat association of non-volant small mammals in Gibe Sheleko National Park,southwestern Ethiopia

The objective of this study was to assess the species composition, abundance and habitat association of non-volant small mammals from grassland, Acacia woodland, farmland, riverine forest, bushland and wooded grassland habitats in Gibe Sheleko National Park, southwestern Ethiopia. Data were collected using 49 Sherman live traps in 70 × 70 m sized girds and visual observations from December 2018 to August 2020. A total of 937 small mammals belonging to 10 species were captured and directly identified. The identified small mammal species and their relative abundance include Arvicanthis niloticus (7.04%), Crocidura olivieri (1.92%), Grammomys dolichurus (3.84%), Lemniscomys striatus (10.25%), Mastomys awashensis (24.55%), Mastomys natalensis (33.83%), Mus tenellus (0.85%), Myomys fumatus (6.30%), Rattus rattus (4.70%) and Stenocephalemys albipes (6.72%). Hystrix cristata, Xerus rutilus and Tachyoryctes splendens were documented through observation. The highest number of species (6) was registered at farmland and wooded grassland followed by bushland and riverine forest (5) and Acacia woodland and grassland (4). Lemniscomys striatus, M. awashensis and M. natalensis were common in all habitats. More, 216 (23.05%) individuals were recorded in farmland, while the least, 111 (11.85%) were recorded in Acacia woodland. The study area has various topographical land settings additional study is needed using different traps.     

Rhizosphere effect of nanoscale zero-valent iron on mycorrhiza-dependent maize assimilation

Rhizosphere effect of nanoscale zero-valent iron (nZVI) is crucial but little reported. Maize seeds were dressed with four nZVI concentrations (0, 1.0, 1.5, 2 g kg⁻¹) and inoculated with arbuscular mycorrhizal fungus (AMF) (Funneliformis mosseae). The SEM images illuminated that excessive nZVI particles (2 g kg⁻¹) were agglomerated on the surface of hyphae and spore, causing severe deformation and inactivation of AMF symbionts and thereafter inhibiting water uptake in maize seedlings. This restrained the scavenging effects of enzymatic (superoxide dismutase, peroxidase) and non-enzymatic compounds (proline & malondialdehyde) on ROS, and leaf photoreduction activity and gas exchange ability (p < 0.05). Interestingly, the inoculation with AMF effectively alleviated above negative effects. In contrast, appropriate dose of nZVI, that is, ≤1.5 g kg⁻¹, can be evenly distributed on the hyphae surface and form the ordered symbionts with AMF. This help massively to enhance hyphae growth and water and nutrient uptake. The enhanced mycorrhizal infection turned to promote rhizosphere symbiont activity and leaf Rubisco and Rubisco activase activity. Light compensation point was massively lowered, which increased photosynthetic carbon supply for AMF symbionts. Particularly, such priming effects were evidently enhanced by drought stress. Our findings provided a novel insight into functional role of nZVI in agriculture and AMF-led green production.     

Matrix synthesis of chiral carboxy derivatives: Aldehyde chiral discrimination due to a two-point coordination of Mg2+

To control stereoselectivity in aldol-like reactions with chiral carbohydrate templates, we studied the interaction between completely protected dialdo compounds and magnesium enediolates of arylacetic acids. Diastereomeric mixtures of the highly functionalized acids obtained were esterified to isolate individual methyl uronates. It was found that all the diastereomeric esters exhibit Cotton effects of the same positive sign in the 220–230 nm region and so possess the same S configuration of the aryl chiral center C(6). Chiral center C(5) configurational assignments were performed using IR and ORD spectroscopy. We separated and specified four pairs of diastereomeric methyl uronates. It follows that the precursory acids have the same 5R*, 6S (major isomers) and 5S*, 6S (minor isomers) configurations. A tentative mechanism for complexation and possible models of Mg²⁺ -protected dialdose intermediate complexes has been proposed. We have concluded that a kind of orbital steering is realized, accompanied by some “tuning” of molecular assembly conditioned by two-point coordination between Mg²⁺ and potential cation-binding sites in the substrate molecules. Thus it has been demonstrated that reasonable diastereo-selectivity can be achieved even through the use of small matrix molecules using rather small functional groups, which do not impose any stringent steric requirements. © 1993 Wiley-Liss, Inc.     

A Rapid Determination of 30 Prohibited Pesticide Residues in Lycii Fructus by UPLC-MS/MS

Prohibited pesticide residues have become one of the main factors affecting the quality and safety of Lycii Fructus, However, rarely studies focus on the rapid determination of these residues. Here, a total of 30 kinds of prohibited pesticide residues were determined by ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-MS/MS) in five different process ways. Pretreatment methods, chromatographic separation and detection conditions in mass spectrometry were all optimized accordingly. Among the five different pretreatment methods, the first and third solid phase extraction failed to provide high recoveries of sulfosulfuron compounds (both lower than 60%). Recovery of chlorphenamidine by the Quick Easy Cheap Effective Rugged and Safe multiresidue method (QuEChERS) was lower than 60%, which did not meet the requirements of trace determination. The concentrations of 30 prohibited pesticides residues treated by straightforward and solid phase extraction showed good linearity in their corresponding ranges, with correlation coefficients over 0.99. The average recoveries of straightforward ranged from 78.13% to 110.9%, while RSD ranged from 1.3% to 16.9%, albeit poor purification was observed. The recovery yield from solid phase extraction was between 67.75% and 103.08% with RSD value from 0.8% to 14.0%, which met the requirements of trace determination, this method has good precision and stability. These results could be employed to other Traditional Chinese Medicines (TCMs) in detecting prohibited pesticide residues.     

Stereoselective sulfate conjugation of isoproterenol in humans: Comparison of hepatic,intestinal, and platelet activity

The stereochemistry of sulfate conjugation of isoproterenol (ISO) was examined with human liver, intestine, and platelets as the phenolsulfotransferase (PST) enzyme source and PAP³⁵S as the cosubstrate. With the hepatic cytosol, two distinct sulfation reactions were identified, a high affinity reaction (K_m 5 to 50 μM) and a low affinity reaction (K_m 360 to 2,900 μM). The efficiency of sulfation (V_max/K_m) for both reactions was 5-fold higher for (+)- than for (?)-ISO. When the hepatic PSTs were resolved by ionexchange chromatography, it could be shown that the high affinity reaction was catalyzed by the monoamine (M) form and the low affinity reaction by the phenol (P) form of PST. Only the high affinity (M form) sulfation was detected in the jejunal cytosol with a V_max/K_m value 6.1-fold higher for (+)- than for (?)-ISO. Finally the platelet, as a potentially useful model tissue, also demonstrated only the high affinity M form reaction with a V_max/K_m value 5.7-fold higher for (+)- than for (?)-ISO. In summary, this study has shown that sulfation of ISO by PSTs in various human tissues is stereoselective and favors the inactive (+)-enantiomer over the active (?)-enantiomer by about 5-fold, a finding which should be considered in the therapeutic use of chiral drugs cleared by sulfate conjugation. © 1993 Wiley-Liss, Inc.     

Extraction,Antioxidant Activity and Identification of Flavonoids from Root Tubers of <i>Kosteletzkya virginica</i>

Kosteletzkya virginica (K. virginica) is used for revegetation of salt-affected coastal tidal flats and as a raw material of biodiesel. K. virginica root tuber, a biowaste with low economic value, is rich in bioactive compounds. This study aimed to extract and identify flavonoids from K. virginica root tubers. The optimal extraction conditions were 1/25 (w/v) solid/liquid ratio, 40% ethanol concentration at 40°C for 60 min. Under these conditions, 65.2 ± 3.7 mg/g total flavonoid content was extracted from the roots, which were collected from salinized soil in late autumn of the third year. Antioxidant activity was evaluated through 1,1-diphenyl-2-picrylhydrazyl, hydroxyl radical, and superoxide anion scavenging assays. The extracted flavonoids exhibited antioxidant activity in a dose-dependent manner. Five flavonoids, glucoliquiritin apioside, licoisoflavone B, 5-methoxy-7,8-diprenyl- flavone, 7,2′-dihydroxy-6,8-dimethyl-4′,5′-methylenedioxyflavan, and 5,7,4′-trihydroxy-3′-methoxy-6,8-di-Cmethylflavanone, were identified by ultra-performance liquid chromatography–tandem mass spectrometry. Our results suggest that the flavonoids of K. virginica root tubers might be potent antioxidants and can be effectively applied as an ingredient in food and natural medicine.     

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.Abbreviations BCECF 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein - TEER transepithelial electrical resistance - LY lucifer yellow CH lithium salt     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.