Protein pattern changes in tomato under in vitro salt stress

The investigation of salt-induced changes in the proteome would highlight important genes because of a high resolution of protein separation by two-dimensional gel electrophoresis (2-DE) and protein identification by mass spectrometry and database search. Tomato (Lycopersicon esculentum Mill.) is a model plant for studying the mechanisms of plant salt tolerance. Seeds of tomato cv. Shirazy were germinated on water-agar medium. After germination, seedlings were transferred to Murashige and Skoog nutrient medium supplemented with 0, 40, 80, 120, and 160 mM NaCl. After 24 days, leaf and root samples were collected for protein extraction and shoot dry weight measurement. Alterations induced in leaf and root proteins under salt stress treatments were studied by one-dimensional SDS-PAGE. Leaf proteins were also analyzed by 2-DE. With increasing salt concentration in the medium, shoot dry weight decreased. SDS-PAGE showed induction of at least five proteins with mol wts of 30, 62, and 75 kD in roots and 38 and 46 kD in leaves. On the 2-DE gel, more than 400 protein spots were reproducibly detected. At least 18 spots showed significant changes under salt stress. Three of them corresponded to new proteins, while six proteins were up-regulated and five proteins were down-regulated by salt stress. In addition, salinity inhibited the synthesis of four leaf proteins. Ten spots were analyzed by matrix-assistant laser desorption/ionization-time of flight (MALDI-TOF), which led to the identification of some proteins, which could play a physiological role under salt stress. The expression of new proteins(enoyl-CoA hydratase, EGF receptor-like protein, salt tolerance protein, phosphoglycerate mutase-like protein, and M2D3.3 protein) under salt stress indicates that tomato leaf cells respond to salt stress by changes in different physiological processes. All identified proteins are somehow related to various salt stress responses, such as cell proliferation. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 526–533. The text was submitted by the authors in English.     

Biotransformation of hydrocortisone by a natural isolate of Nostoc muscorum

Hydrocortisone was converted in the culture of an isolated strain of the cyanobacterium Nostoc muscorum PTCC 1636 into some androstane and pregnane derivatives. The microorganism was, isolated during a screening program from soil samples collected from paddy fields of north of Iran. The bioproducts obtained were purified using chromatographic methods and identified as 11beta-hydroxytestosterone, 11beta-hydroxyandrost-4-en-3,17-dione and 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one on the basis of their spectroscopic features.     

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman–Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P4_1, with R_work/R_free values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme–inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.     

Rate of recalls in congenital hypothyroidism based upon a regional survey in Isfahan, Iran, using serum T4 and TSH analyses: comparison of two different recall methods

AIMS: To evaluate and compare the recall rate in congenital hypothyroidism screening project in Isfahan, first using an approach involving measures of both TSH and T4 and then using TSH alone. METHODS: From June 2002 to January 2005, serum TSH and T4 level of referred neonates were measured at 3rd to 7th day of birth through venous sampling. If neonates' serum TSH was >20 mIU/l or T4 was <6.5 microg/dl by the first protocol, or TSH was >20 mIU/l by the second protocol, they were recalled. TSH and T4 were measured using an immunoradiometric assay and radioimmunoassay, respectively. Neonates with TSH > 10 and T4 < 6.5 on their second measurement were considered as congenitally hypothyroid. RESULTS: Serum T4 and TSH of 29,425 neonates by first and 57,235 neonates by second recall approach were measured. Recall rate was higher in the first protocol (2.2% vs. 0.6%, p < 0.05). Most of the recalled neonates in the first protocol were recalled for low T4 level (p < 0.05). The prevalence of CH was 1 in 350 livebirths. CONCLUSION: Although the recall rate was in the acceptable range by either approach, the TSH alone protocol seems to be a more sensitive and practical approach with the least recall burden and considering the high prevalence of CH in our region merit adaptation of widespread screening for CH using TSH measurements from heel stab blood spotted on filter paper.     

Carbohydrate-active enzymes involved in the secondary cell wall biogenesis in hybrid aspen

Wood formation is a fundamental biological process with significant economic interest. While lignin biosynthesis is currently relatively well understood, the pathways leading to the synthesis of the key structural carbohydrates in wood fibers remain obscure. We have used a functional genomics approach to identify enzymes involved in carbohydrate biosynthesis and remodeling during xylem development in the hybrid aspen Populus tremula x tremuloides. Microarrays containing cDNA clones from different tissue-specific libraries were hybridized with probes obtained from narrow tissue sections prepared by cryosectioning of the developing xylem. Bioinformatic analyses using the sensitive tools developed for carbohydrate-active enzymes allowed the identification of 25 xylem-specific glycosyltransferases belonging to the Carbohydrate-Active EnZYme families GT2, GT8, GT14, GT31, GT43, GT47, and GT61 and nine glycosidases (or transglycosidases) belonging to the Carbohydrate-Active EnZYme families GH9, GH10, GH16, GH17, GH19, GH28, GH35, and GH51. While no genes encoding either polysaccharide lyases or carbohydrate esterases were found among the secondary wall-specific genes, one putative O-acetyltransferase was identified. These wood-specific enzyme genes constitute a valuable resource for future development of engineered fibers with improved performance in different applications.     

Molecular cloning and characterization of a cDNA encoding poplar UDP-glucose dehydrogenase,a key gene of hemicellulose/pectin formation

Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of newly formed cell walls. A cDNA clone (Ugdh) corresponding to UGDH was isolated from a cDNA library prepared from cambial zone of poplar (Populus tremula x tremuloides). Within the 1824-nucleotide (nt)-long clone, an open reading frame encoded a protein of 481 amino acids (aa), with a calculated molecular weight of 53.1 kDa. The derived aa sequence showed 90% and 63% identity with UGDHs from soybean and bovine liver, respectively, and had highly conserved aa motifs believed to be of importance for nt binding and catalytic efficiency. In poplar, the Ugdh corresponds to one or two genes, as found by genomic Southern analysis. The gene was expressed predominantly in differentiating xylem and young leaves, with little expression in the phloem zone of the stem. The expression pattern matched that of UGDH protein, as found by immunoblotting. In leaves, the Ugdh expression was upregulated by a short-term feeding with sucrose, sorbitol and polyethylene glycol, and this effect was to some extent mimicked by light exposure. The data suggest that Ugdh is regulated via an osmoticum-dependent pathway, possibly related to the availability of osmotically active carbohydrate precursors to UDP-glucose, a substrate of UGDH.