Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases

Class 2 aminoacyl-tRNA synthetases, which include the enzymes for alanine, aspartic acid, asparagine, glycine, histidine, lysine, phenylalanine, proline, serine and threonine, are characterised by three distinct sequence motifs 1,2 and 3 (reference 1). The structural and evolutionary relatedness of these ten enzymes are examined using alignments of primary sequences from prokaryotic and eukaryotic sources and the known three dimensional structure of seryl-tRNA synthetase from E. coli. It is shown that motif 1 forms part of the dimer interface of seryl-tRNA synthetase and motifs 2 and 3 part of the putative active site. It is further shown that the seven alpha 2 dimeric synthetases can be subdivided into class 2a (proline, threonine, histidine and serine) and class 2b (aspartic acid, asparagine and lysine), each subclass sharing several important characteristic sequence motifs in addition to those characteristic of class 2 enzymes in general. The alpha 2 beta 2 tetrameric enzymes (for glycine and phenylalanine) show certain special features in common as well as some of the class 2b motifs. In the alanyl-tRNA synthetase only motif 3 and possibly motif 2 can be identified. The sequence alignments suggest that the catalytic domain of other class 2 synthetases should resemble the antiparallel domain found in seryl-tRNA synthetase. Predictions are made about the sequence location of certain important helices and beta-strands in this domain as well as suggestions concerning which residues are important in ATP and amino acid binding. Strong homologies are found in the N-terminal extensions of class 2b synthetases and in the C-terminal extensions of class 2a synthetases suggesting that these putative tRNA binding domains have been added at a later stage in evolution to the catalytic domain.     

Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence.

Although partial or complete three-dimensional structures are known for three Class I aminoacyl-tRNA synthetases, the amino acid-binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl-tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl-tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.-P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449-457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300-fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the "signature sequence" of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl-tRNA synthetases and, in the known structures, it is in the nucleotide-binding fold as part of a loop between the end of a beta-strand and the start of an alpha-helix. The phenotype of the mutant and the stability and affinity for methionine of the wild-type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C-terminus of the signature sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular distribution and properties of rabbit liver aminoacyl-tRNA synthetases under myocardial ischemia

Subcellular distribution of aminoacyl-tRNA synthetase activities has been studied in normal rabbit liver and under experimental myocardial ischemia (EMI). An increase in the activity of a number of aminoacyl-tRNA synthetases in postmitochondrial and postribosomal supernatants from rabbit liver has been determined 12 hr after EMI. Gel chromatography of the postribosomal supernatant on Sepharose 6B shows that aminoacyl-tRNA synthetase activities are distributed among the fractions with M_r 1.82×10⁶, 0.84×10⁶ (high-M_r aminoacyl-tRNA synthetase complexes) and 0.12–0.35×10⁶. In the case of EMI aminoacyl-tRNA synthetase activities are partly redistributed from the 1.82×10⁶ complex into the 0.84×10⁶ complex. The catalytic properties of both free and complex leucyl-tRNA synthetases have been compared. K_M for all the substrates are the values of the same order in norm and under EMI. A decrease in some aminoacyl-tRNA synthetase activities associated with polyribosomes has been observed 12 hr after EMI. The interaction of aminoacyl-tRNA synthetases with polyribosomes stimulates the catalytic activity of some enzymes and protects them from heat inactivationin vitro. It is assumed that the changes in association of aminoacyl-tRNA synthetases with high-M_r complexes and compartmentalization of these enzymes on polyribosomes may be related to the alteration of protein biosynthesis under myocardial ischemia.     

Leukotriene D4-induced adhesion of Caco-2 cells is mediated by prostaglandin E2 and upregulation of alpha2beta1-integrin

Cell-cell and extracellular matrix adhesions play important roles in the progression of cancer. We investigated the involvement of the inflammatory mediator leukotriene D4 (LTD4) in the regulation of cell-matrix adhesion of colon cancer (Caco-2) cells. We observed that LTD4 acted via its CysLT1 receptor in these cells to induce increased adhesion to collagen I. LTD4 also enhanced the activation and expression of alpha2beta1-integrins on the cell surface, which we found to be responsible for mediating the increased adhesion to collagen I. LTD4 simultaneously augmented expression of the prostaglandin-generating enzyme cyclooxygenase-2 (COX-2) and increased prostaglandin E2 (PGE2) production in Caco-2 cells. The adhesive capacity of the Caco-2 cells was reduced by specific inhibition of COX-2 and was subsequently restored by PGE2, but not by LTD4. A selective PGE2 receptor antagonist abolished the increased adhesion and the augmented alpha2beta1-integrin expression induced by both PGE2 and LTD4. Summarizing, the inflammatory mediator LTD4 regulates the adhesive properties and migration of the Caco-2 cell line by upregulating COX-2 and stimulating PGE2-induced expression of alpha2beta1-integrins. This suggests that inflammatory mediators such as LTD4 can be involved in the dissemination and survival of colon cancer cells.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Integrin αvβ3, metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect

Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin α_vβ₃, evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the α_vβ₃ blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin α_vβ₃ interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin α_vβ₃, uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin α_vβ₃ and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.     

A common evolutionary origin of two elementary enzyme folds

The (beta alpha)(8)-barrel is the most frequent and most versatile fold among enzymes [H?cker et al., Curr. Opin. Biotechnol. 12 (2001) 376-381; Wierenga, FEBS Lett. 492 (2001) 193-198]. Structural and functional evidence suggests that (beta alpha)(8)-barrels evolved from an ancestral half-barrel, which consisted of four (beta alpha) units stabilized by dimerization [Lang et al., Science 289 (2000) 1546-550; H?cker et al., Nat. Struct. Biol. 8 (2001) 32-36; Gerlt and Babbitt, Nat. Struct. Biol. 8 (2001) 5-7]. Here, by performing a comprehensive database search, we detect a striking and unexpected structural and amino acid sequence similarity between (beta alpha)(4) half-barrels and members of the (beta alpha)(5) flavodoxin-like fold. These findings provoke the hypothesis that a large fraction of the modern-day enzymes evolved from a basic structural building block, which can be identified by a combination of sequence and structural analyses.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma application to nutritional studies

A simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D₂ and D₃ in human plasma was developed. Plasma samples were deproteinizated and applied to a Bond Elut C₁₈ OH cartridge to separate 25-hydroxyvitamin D (25-OH-D) and 1α-25-dihydroxyvitamin D [1,25(OH)₂D] fractions. The 25-OH-D fraction was purified by a Bond Elut C₁₈ cartridge and 25-OH-D₂ and 25-OH-D₃ were assayed by HPLC using a Zorbax SIL column. The 1,25(OH)₂D fraction obtained above was subsequently applied to HPLC using a Zorbax SIL column to separate 1,25(OH)₂D₂ and 1,25(OH)₂D₃ fractions which were determined by a radioreceptor assay (RRA) using calf thymus receptor. The method was applied to nutritional studies.     

Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein

Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to alphaIIbbeta3 integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS-PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an alphaIIbbeta3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and alphaIIbbeta3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the alphaIIbbeta3 integrin is involved.     

Beta-1 integrins mediate substrate dependent effects of 1α,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. β₁ integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1α,25(OH)₂D₃ in a surface-dependent manner. To determine if β₁ has a role in mediating osteoblast response, we silenced β₁ expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human β₁ to block ligand binding. β₁-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β₁, prostaglandin E₂, and osteoprotegerin in comparison with control cells. Moreover, β₁-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)₂D₃. Anti β₁ antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1α,25(OH)₂D₃ on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β₁ plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)₂D₃. The results also show that β₁ mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)₂D₃.     

Characterization of the oligosaccharide component of alpha3beta1 integrin from human bladder carcinoma cell line T24 and its role in adhesion and migration

Malignant transformation is highly associated with altered expression of cell surface N-linked oligosaccharides. These changes concern integrins, a family of cell surface glycoproteins involved in the attachment and migration of cells on various extracellular matrix proteins. The integrin alpha3beta1 is particularly interesting because of its role in migration and invasion of several types of metastatic tumours. In this study, alpha3beta1 from human bladder T24 carcinoma cells was purified and treated with peptide N-glycosidase F. Then the N-glycans of the alpha3 and beta1 subunits were characterized using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In alpha3beta1 integrin the presence of high-mannose, hybrid and predominantly complex type N-oligosaccharides was shown. Unlike to normal epithelium cells, in both subunits of alpha3beta1 integrin from cancer cells, the sialylated tetraantennary complex type glycan Hex7HexNAc6FucSia4 was present. In a direct ligand binding assay, desialylated alpha3beta1 integrin exhibited significantly higher fibronectin-binding capability than untreated integrin, providing evidence that sialic acids play a direct role in ligand-receptor interaction. Moreover, alpha3beta1 integrin was shown to take part in T24 cell migration on fibronectin: anti-alpha3 antibodies induced ca 30% inhibition of wound closure. Treatment of T24 cells with swainsonine reduced the rate of bladder carcinoma cell migration by 16%, indicating the role of beta1,6 branched complex type glycans in this process. Our data show that alpha3beta1 integrin function may be altered by glycosylation, that both subunits contribute to these changes, and that glycosylation may be considered a newly found mechanism in the regulation of integrin function.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

EFFECT OF ULCERATIVE COLITIS ACTIVITY ON PLASMA CONCENTRATION OF TRANSFORMING GROWTH FACTOR β1

Enhanced expression of transforming growth factor-β₁(TGF-β₁) demonstrated in human colonic mucosa of patients with ulcerative colitis (UC), indicates its possible significance in the pathogenesis of this disease. The aim of this study was to evaluate plasma TGF-β₁concentration in patients with different degrees of colonic mucosal injury, as a possible indicator of ulcerative colitis activity. TGF-β₁concentration was measured with an enzyme immunoassay (EIA) in plasma of 45 patients with endoscopically confirmed UC. Values observed in UC patients (40.5±15.9 ng/ml) were significantly higher than in healthy people (18.3±11.6 ng/ml) and higher than in patients with irritable colon syndrome (ICS), (20.5±13.6 ng/ml). The highest plasma TGF-β₁(58.6±112.1 ng/ml) was in patients with the severe UC course. TGF-β₁level analysed in all UC patients revealed significant positive correlation with scored degree of mucosal injury (r=0.396;P<0.01). Among other possible laboratory markers of the disease activity, only C-reactive protein concentration demonstrated significant correlation. Enhanced production of TGF-β₁can be related to inflammation activity. Measurement of plasma TGF-β₁may be considered as a biomarker of the disease activity.     

Determination of isoprostaglandin F2α type III in human urine by gas chromatography–electronic impact mass spectrometry. Comparison with enzyme immunoassay

F₂-Isoprostanes are stable lipid peroxidation products of arachidonic acid, the quantification of which provides an index of oxidative stress in vivo. We describe a method for analysing isoprostaglandin F_2α type III (15-F_2t-IsoP) in biological fluids. The method involves solid-phase extraction on octadecyl endcapped and aminopropyl cartridges. After conversion to trimethylsilyl ester trimethylsilyl ether derivatives, isoprostaglandin F_2α type III is analysed by mass spectrometry, operated in electronic impact selected ion monitoring mode. We have compared enzyme immunoassay (EIA; Cayman, Ann Arbor, MI, USA) to this method with 30 human urine aliquots following the same extraction procedure in order to determine the agreement between both methods. Isoprostaglandin F_2α type III concentrations determined with gas chromatography–mass spectrometry (GC–MS) did not agree with those determined with EIA. Our results suggest that GC–MS and EIA do not measure the same compounds. As a consequence, comparison of clinical results using GC–MS and EIA should be avoided.     

Synthesis and biological evaluation of a 3-positon epimer of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71)

1α,25-Dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃ (ED-71), an analog of active vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], possesses a hydroxypropoxy substituent at the 2β-position of 1,25(OH)₂D₃. ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)₂D₃. Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)₂D₃, an epimer of 1,25(OH)₂D₃ at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)₂D₃ at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D₃ analogs were found to be 1,25(OH)₂D₃ > ED-71 ≥ 3-epi-1,25(OH)₂D₃  3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.     

A facile synthesis, structure, and antimicrobial evaluation of novel 4-arylhydrazono-5-trifluoromethyl-2,4-dihydropyrazol-3-ones, their N- and N,O-bis-β-d-glucosides

Synthesis of some novel 4-arylhydrazono-5-trifluoromethyl-2,4-dihydropyrazol-3-ones, their N- and N,O-bis- β-d-glucosides is described. Antimicrobial evaluation of eight selected compounds against Aspergillus fumigatus RCMB 002008 (1), Penicillium italicum RCMB 001018 (1), Syncephalastrum racemosum RCMB 016001, Candida albicans RCMB 005003, Staphylococcus aureus RCMB 106-001 (1), Pseudomonas aeruginosa RCMB 102-002, Bacillus subtilis RCMB 101-001, and Escherichia coli RCMB 103-001 has been achieved. The screening results indicated that all the tested compounds exhibited different inhibitory effects against five to seven different organisms of the eight test organisms.     

Biosynthesis and catabolism of prostaglandin F2alpha (PGF2alpha) are controlled by progesterone in the rat uterus during pregnancy

Myometrial quiescence is a key factor in all species to accomplish a successful gestation. PGs play a crucial role in mediating parturition events, and their synthesis and metabolism are regulated by cyclooxygenases (COXs) and NAD(+)-dependent 15-hydroxy-PG dehydrogenase (PGDH), respectively. Progesterone (P(4)) is the hormone responsible for maintaining uterine smooth muscle quiescence during pregnancy. In this work, we have studied the effect of P(4) on the activity of COXs and PGDH, the uterine enzymes involved in the biosynthesis and metabolism of prostanoids in the rat. We found that during pregnancy PGF(2alpha) production and also protein levels of COX-1 and COX-2 were decreased. The exogenous administration of P(4) significantly inhibited the uterine production of PGF(2alpha) and also the protein level of COX-2. PGF(2alpha), metabolism was assessed by PGDH activity, which resulted high during pregnancy and increased as a result of P(4) administration. These results indicate that PGs levels were negatively modulated by P(4), which could be exerting its effect by increasing PGs metabolism through stimulation on PGDH activity and an inhibition on COX and that is a major mechanism for maintain uterine quiescence in pregnancy.     

Purification, biochemical characterisation and partial primary structure of a new α-amylase inhibitor from Secale cereale (rye)

Plant α-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the α-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13 756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional α-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus α-amylases was observed. The inhibitor is more effective against insect α-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional α-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.