Influence of Acidity on Growth and Biochemistry of Pennisetum clandestinum

Hydroponics were used to study the impact of acidity on growth, nutritive properties and metabolic changes in kikuyu grass (Pennisetum clandestinum Hochst). Four treatments (pH 6.0, 5.0, 4.0, and 3.0) were compared for effects on biomass, leaf and root length, crude protein, amino acid content and key enzymes of sugar metabolism. Reduction in biomass, root and leaf length, amino acid contents, glucose-6-phosphate dehydrogenase (G6PDH) and pyruvate kinase (PK) content was observed only at pH 3.0, in association with increased leaf proline content. Kikuyu grass is able to grow normally under mild acidity (down to at least pH 4.0).     

Osmolyte accumulation in different rape genotypes under sodium chloride salinity

Physiological mechanisms of two rape (Brassica napus L.) genotype adaptation to chlorine salinity were investigated. The plants of two cultivars (Olga and Westar) differing in salt tolerance were grown in the pots filled with Perlite on the Hoagland and Snyder’s medium under controlled conditions. At a stage of 3–4 true leaves, the plants experienced 7-day-long salinity induced by a single addition of NaCl to the nutrient medium in order to attain desired final salt concentration (from 50 to 400 mM). The obtained results showed that a greater salt tolerance of cv. Olga plants (as compared with cv. Westar) could be accounted for by a capability of their root cells to uptake water under high salinity (300–400 mM NaCl), which is evident from a greater content of water in the tissues of cv. Olga. This was ensured by a sharp fall of the osmotic potential of the cellular contents (down to −2.3 MPa) at a low water potential of nutrient solution owing to more active uptake of Na⁺ (57–61 μeq/g fr wt) and K⁺ (210–270 μeq/g fr wt) as well as active accumulation of proline (30–50 μmol/g fr wt). The latter is caused by a reduced activity of proline dehydrogenase and retarded degradation of this osmolyte. It is important that, in contrast to less tolerant genotype, the rape plants of salt-resistant cultivar were able to maintain the K⁺/Na⁺ ratio at a rather high level at salinity of different degree, which made it possible to preserve ionic homeostasis under adverse conditions. Original Russian Text ? A.M. Mokhamed, G.N. Raldugina, V.P. Kholodova, Vl.V. Kuznetsov, 2006, published in Fiziologiya Rastenii, 2006, Vol. 53, No. 5, pp. 732–738.     

The planar conformation of a strained proline ring: a QM/MM study

QM and QM/MM energy calculations have been carried out on an atomic resolution structure of liganded triosephosphate isomerase (TIM) that has an active site proline (Pro168) in a planar conformation. The origin of the planarity of this proline has been identified. Steric interactions between the atoms of the proline ring and a tyrosine ring (Tyr166) on one side of the proline prevent the ring from adopting the up pucker (chi1 is approximately -30 degrees), while the side chain of a nearby alanine (Ala171) forbids the down pucker (chi1 is approximately +30 degrees). To obtain a proline conformation that is in agreement with the experimentally observed planar state, a quantum system of sufficient size is required and should at least include the nearby side chains of Tyr166, Ala171, and Glu129 to provide enough stabilization. It is argued that the current force fields for structure optimization do not describe strained protein fragments correctly. The proline is part of a catalytic loop that closes upon ligand binding. Comparison of the proline conformation in different TIM X-ray structures, indicates that in the closed conformation of TIM the proline is planar or nearly planar, while in the open conformation it is down puckered. This suggests that the planarity possibly plays a role in the overall catalytic cycle of TIM, presumable acting as a reservoir of energy that becomes available upon loop opening.     

Alanine scanning mutagenesis of Abeta(1-40) amyloid fibril stability

We describe here an alanine scanning mutational analysis of the Abeta(1-40) amyloid fibril monitored by fibril elongation thermodynamics derived from critical concentration values for fibril growth. Alanine replacement of most residues in the amyloid core region, residues 15-36, leads to destabilization of the elongation step, compared to wild-type, by about 1kcal/mol, consistent with a major role for hydrophobic packing in Abeta(1-40) fibril assembly. Where comparisons are possible, the destabilizing effects of Ala replacements are generally in very good agreement with the effects of Ala replacements of the same amino acid residues in an element of parallel beta-sheet in the small, globular protein Gbeta1. We utilize these Ala-WT DeltaDeltaG values to filter previously described Pro-WT DeltaDeltaG values, creating Pro-Ala DeltaDeltaG values that specifically assess the sensitivity of a sequence position, in the structural context of the Abeta fibril, to replacement by proline. The results provide a conservative view of the energetics of Abeta(1-40) fibril structure, indicating that positions 18-21, 25-26, and 32-33 within amyloid structure are particularly sensitive to the main-chain disrupting effects of Pro replacements. In contrast, residues 14-17, 22, 24, 27-31, and 34-39 are relatively insensitive to Pro replacements; most N-terminal residues were not tested. The results are discussed in terms of amyloid fibril structure and folding energetics, in particular focusing on how the data compare to those from other structural studies of Abeta(1-40) amyloid fibrils grown in phosphate-buffered saline at 37 degrees C under unstirred ("quiescent") conditions.     

Analysis of the CD2 and spliceosomal Sm B/B' polyproline-arginine motifs defined by a monoclonal antibody using a phage-displayed random peptide library

The cytoplasmic region of the CD2 receptor of lymphocytes contains proline-rich motifs, which are involved in T cell activation and interleukin-2 production. An intracellular CD2 binding protein, CD2BP2, interacts with two tandem PPPPGHR segments of the CD2 tail. CD2BP2 contains a GYF (glycine-tyrosine-phenylalanine) domain that confers binding to these proline-rich sequences. Monoclonal antibody 3E10 that was previously raised against a peptide containing the CD2 PPPPGHR segment reacts with the native CD2 molecule and spliceosomal Sm B/B' proteins. To identify the exact epitope on the CD2 peptide recognized by 3E10, a phage-displayed combinatorial peptide library was used. Analysis of the selected clones revealed that the mAb 3E10 binds preferentially to the motif PxxPPGxR. Experiments using amino acid substitutions with synthetic peptides confirmed the reactivity of mAb 3E10 with this motif. In addition, we show that several similarities exist between this motif and the CD2BP2-GFY recognition motif PPGxR/K. Binding of antibody 3E10 indicates some degree of degeneracy, which is consistent with its ability to recognize structurally related polyproline-arginine motifs found in intracellular proteins including Sm B/B' proteins and other RNA binding proteins. Thus, mAb 3E10 can be used to specifically identify a sub-class of proline-rich motifs, and as such can be used to study the potential role of these proline-rich sequences in mediating protein-protein interactions.     

The molecular and biochemical characteristics of proline iminopeptidase from rye seedlings (Secale cereale L.)

A proline iminopeptidase (EC. 3.4.11.5) was isolated from shoots of 3 day old seedlings. The purification procedure consisted of 5 steps: acid precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on Sepharose CL 6B, twice repeated hydrophoic chromatography on Phenyl-Sepharose HP. The enzyme was purified 404.8-fold, with the specific activity of 8.5 units mg⁻¹ of protein with recovery yield of 3%. The purified enzyme had a molecular mass of 225 kDa estimated by gel filtration and 55.4 kDa by SDS PAGE. This indicates that native enzyme is composed of four subunits. The enzyme was specific for proline β-naphtylamide among various amino acid β-naphtylamides. An optimal activity was observed at 37 °C at pH 7.75. The enzyme was thermostable up to 37 °C for 30 min. The enzyme was strongly inhibited by pHMB, E-64, heavy metal ions and partially by PMSF, DFP. The results suggest that cysteine and serine residues may participate in the enzyme activity.     

Water relations, activities of antioxidants, ethylene evolution and membrane integrity of pigeonpea roots as affected by soil moisture

The plants of pigeonpea (Cajanus cajan L.) cv. H77-216 were subjected to moderate [soil moisture content (SMC) = 7.3 ± 0.5 %] and severe (SMC = 4.3 ± 0.5 %) drought by withholding the irrigation at vegetative stage (45 d after sowing). The control plants were maintained at SMC of 11.0 ± 0.5 %. Half of the stressed plants were re-irrigated and their recovery was studied after 2 d. Leaf water potential, osmotic potential, and relative water content of leaf and root decreased significantly while a sharp rise in proline and total soluble sugars contents were noticed. Drought induced a significant increase in 1-aminocyclopropane 1-carboxylic acid (ACC) content and ACC oxidase activity which caused a considerable increase in ethylene evolution. Malondialdehyde content and relative stress injury were increased under drought whereas reverse was true for ascorbic acid content. The membrane integrity of roots decreased during stress and recovered on rehydration. The specific activity of total superoxide dismutase, ascorbate peroxidase, glutathione reductase, and glutathione transferase decreased to 37 – 78 %, 17 – 62 %, 29 – 36 % and 57 – 79 % at moderate and severe drought, respectively. The increase in activity of catalase and peroxidase could not overcome the accumulation of H₂O₂ content in the roots.     

Associations between two novel rSNPs in 5'-flanking region of the bovine casein gene cluster and milk performance traits

Ten evolutionary conservative sequences with high identity level to homological sequences in other mammal species were revealed in 5'-flanking region of casein's genes cluster. Five novel SNPs located inside of the evolutionary conservative regions were identified. The binding sites were revealed to be present in one allelic variant of four detected SNPs. So these SNPs were considered as rSNPs. Significant differences of allelic frequencies were revealed between beef cow's group and dairy cow's group in two rSNPs (NCE4, NCE7, p<0.001). Different alleles of those two rSNPs were shown to be associated with some milk performance traits in Black-and-White Holstein dairy cows. Significant difference of protein percentage has been found between cows with G/G and A/A genotypes (P<0.05) and A/G and A/A genotypes (P<0.05) for NCE4 polymorphism. The groups of animals with genotypes G/G and A/G for NCE7 polymorphism were significantly different in milk yield at the first lactation (kg) (P<0.01), milk fat yield (kg) (P<0.05) and milk protein yield (kg) (P<0.01). For the last trait the difference was significant also between cows with genotypes G/G and A/A for rSNP NCE7 (P<0.05).     

Absolute Configuration Determination and Convenient Asymmetric Synthesis of cis-3-(9-Anthryl)cyclohexanol with Proline as a Catalyst

cis‐(3R)‐(9‐anthryl) derivative of cyclohexanol was conveniently obtained in enantiomerically pure form from 2‐cyclohexenone using asymmetric Michael addition of anthrone catalyzed by l ‐proline in a key step. The absolute configuration of the addition product was unequivocally determined by means of electronic circular dichroism measurements combined with calculation of the circular dichroism spectrum by using a density functional theory method. Chirality 24:833–839, 2012. © 2012 Wiley Periodicals, Inc.     

Isoform identification, recombinant production and characterization of the allergen lipid transfer protein 1 from pear (Pyr c 3)

Non-specific lipid transfer proteins belonging to LTP1 family represent the most important allergens for non pollen-related allergies to Rosaceae fruits in the Mediterranean area. Peach LTP1 (Pru p 3) is a major allergen and is considered the prototypic allergenic LTP. On the contrary, pear allergy without pollinosis seems to be under-reported when compared to other Rosaceae fruits suggesting that the as-yet-uncharacterized pear LTP1 (Pyr c 3) has in vivo a low allergenicity. We report here on the identification of four cDNAs encoding for LTP1 in pear fruits. The two isoforms exhibiting amino acid sequences most similar to those of peach and apple homologues were obtained as recombinant proteins. Such isoforms exhibited CD spectra and lipid binding ability typical of LTP1 family. Moreover, pear LTP1 mRNA was mainly found in the peel, as previously shown for other Rosaceae fruits. By means of IgE ELISA assays a considerable immunoreactivity of these proteins to LTP-sensitive patient sera was detected, even though allergic reactions after ingestion of pear were not reported in the clinical history of the patients. Finally, the abundance of LTP1 in protein extracts from pear peel, in which LTP1 from Rosaceae fruits is mainly confined, was estimated to be much lower as compared to peach peel. Our data suggest that the two isoforms of pear LTP1 characterized in this study possess biochemical features and IgE-binding ability similar to allergenic LTPs. Their low concentrations in pear might be the cause of the low frequency of LTP-mediated pear allergy.