A simple strategy for the purification of large thermophilic proteins overexpressed in mesophilic microorganisms: application to multimeric enzymes from Thermus sp. strain T2 expressed in Escherichia coli

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an alpha-galactosidase and a beta-galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.     

Reversible immobilization of glucoamylase by ionic adsorption on sepabeads coated with polyethyleneimine

Glucoamylase (GA) from Aspergillus niger was immobilized via ionic adsorption onto DEAE-agarose, Q1A-Sepabeads, and Sepabeads EC-EP3 supports coated with polyethyleneimine (PEI). After optimization of the immobilization conditions (pH, polymer size), it was observed that the adsorption strength was much higher in PEI-Sepabeads than in Q1A-Sepabeads or DEAE-supports, requiring very high ionic strength to remove glucoamylase from the PEI-supports (e.g., 1 M NaCl at pH 5.5). Thermal stability and optimal temperature was marginally improved by this immobilization. Recovered activity depended on the substrate used, maltose or starch, except when very low loading was used. The optimization of the loading allowed the preparation of derivatives with 750 IU/g in the hydrolysis of starch, preserving a high percentage of immobilized activity (around 50%).     

An Lsm2-Lsm7 complex in Saccharomyces cerevisiae associates with the small nucleolar RNA snR5

Sm-like (Lsm) proteins function in a variety of RNA-processing events. In yeast, the Lsm2-Lsm8 complex binds and stabilizes the spliceosomal U6 snRNA, whereas the Lsm1-Lsm7 complex functions in mRNA decay. Here we report that a third Lsm complex, consisting of Lsm2-Lsm7 proteins, associates with snR5, a box H/ACA snoRNA that functions to guide site-specific pseudouridylation of rRNA. Experiments in which the binding of Lsm proteins to snR5 was reconstituted in vitro reveal that the 3' end of snR5 is critical for Lsm protein recognition. Glycerol gradient sedimentation and sequential immunoprecipitation experiments suggest that the Lsm protein-snR5 complex is partly distinct from the complex formed by snR5 RNA with the box H/ACA proteins Gar1p and Nhp2p. Consistent with a separate complex, Lsm proteins are not required for the function of snR5 in pseudouridylation of rRNA. We demonstrate that in addition to their known nuclear and cytoplasmic locations, Lsm proteins are present in nucleoli. Taken together with previous findings that a small fraction of pre-RNase P RNA associates with Lsm2-Lsm7, our experiments suggest that an Lsm2-Lsm7 protein complex resides in nucleoli, contributing to the biogenesis or function of specific snoRNAs.     

A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis suaveolens L.) inhibits proteases from the larger grain borer Prostephanus truncatus (Coleoptera: Bostrichidae)

A novel trypsin inhibitor purified from chan seeds (Hyptis suaveolens, Lamiaceae) was purified and characterized. Its apparent molecular mass was 8700 Da with an isoelectric point of 3.4. Its N-terminal sequence showed a high content of acidic amino acids (seven out of 18 residues). Its inhibitory activity was potent toward all trypsin-like proteases extracted from the gut of the insect Prostephanus truncatus (Coleoptera: Bostrichidae), a very important pest of maize. This activity was highly specific, because among proteases from seven different insects, only those from P. truncatus and Manduca sexta (Lepidoptera: Sphingidae) were inhibited. This inhibitor has potential to enhance the defense mechanism of maize against the attack of P. truncatus.     

Auxin pulses and a synergistic interaction between polyamines and ethylene inhibitors improve adventitious regeneration from apricot leaves and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of leaf tissues

The effect, on adventitious regeneration from apricot leaf explants and transformation of leaf tissues, of auxins pulses with NAA and 2, 4-D was tested. Addition of the polyamines putrescine and spermidine to the regeneration medium, alone or in combination with the ethylene inhibitors silver thiosulphate and aminoethoxyvinylglycine, were also tested to design a procedure that improved transformation efficiency. Spermidine at 2 mM in combination with 0.5 M aminoethoxyvinylglycine and four-day pulses with two different concentrations of 2, 4-D increased significantly shoot regeneration. Spermidine at the same concentration but in combination with 60 M silver thiosulphate and four-day pulses with 9 M 2, 4-D also increased stable transformation events and GFP-expressing calluses probably by inducing a larger amount of dividing cells where Agrobacterium transferred its T-DNA. Since regeneration from apricot leaves occurs mostly from developing calluses, it is important to obtain many GFP-expressing calluses and, given that transformation efficiencies (number of transformed shoots per total number of explants) in woody plants are generally very low, approaches that allow the optimization of T-DNA transfer and total number of transformed cells obtained, will improve probabilities of obtaining transformed shoots.     

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports

In this work, we have used supports activated with m-amino-phenylboronic groups to “reversibly” immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.     

Regulation of the translation initiation factor eIF4F by multiple mechanisms in human cytomegalovirus-infected cells

As a viral opportunistic pathogen associated with serious disease among the immunocompromised and congenital defects in newborns, human cytomegalovirus (HCMV) must engage the translational machinery within its host cell to synthesize the viral proteins required for its productive growth. However, unlike many viruses, HCMV does not suppress the translation of host polypeptides. Here, we examine how HCMV regulates the cellular cap recognition complex eIF4F, a critical component of the cellular translation initiation apparatus that recruits the 40S ribosome to the 5' end of the mRNA. This study establishes that the cap binding protein eIF4E, together with the translational repressor 4E-BP1, are both phosphorylated early in the productive viral growth cycle and that the activity of the cellular eIF4E kinase, mnk, is critical for efficient viral replication. Furthermore, HCMV replication also induces an increase in the overall abundance of eIF4F components and promotes assembly of eIF4F complexes. Notably, increasing the abundance of select eIF4F core components and associated factors alters the ratio of active eIF4F complexes in relation to the 4E-BP1 translational repressor, illustrating a new strategy through which members of the herpesvirus family enhance eIF4F activity during their replicative cycle.     

Vacuolating cytotoxin A is associated with increased thrombin generation in gastric mucosa

BACKGROUND: Activation of the coagulation system is a critical response for both the repair of tissue injury and the host defense against microbial pathogens. Activation of the coagulation cascade culminates with the generation of thrombin. In vitro studies have shown that thrombin protects gastric epithelial cells from injury. The present study was undertaken to assess in vivo the relationship between gastric intramucosal generation of thrombin and Helicobacter pylori infection. MATERIALS AND METHODS: This study comprised 59 patients with gastroduodenal disorders. There were 27 patients with H. pylori infection (Hp+), 14 without it (Hp-), and 18 patients with cured H. pylori infection (Hp c). The gastric intramucosal concentrations of thrombin-antithrombin complex (TAT), epidermal growth factor (EGF), prostaglandin E2 (PGE2), and vacuolating cytotoxin A (VacA) were measured by specific immunoassays. RESULTS: The level of TAT was significantly increased in patients with Hp+ compared to Hp- and Hp c. The levels of TAT, EGF and PGE2 were higher in VacA (+) patients than in those with VacA (-). VacA induced significant expression of tissue factor in gastric epithelial cells in vitro. The gastric intramucosal level of VacA antigen was proportionally and significantly correlated with TAT, EGF and PGE2 in Hp+ patients. The level of TAT was proportionally and significantly correlated with EGF in Hp+ patients but not in Hp- and HP c patients. CONCLUSIONS: These results showed that VacA produced by H. pylori is associated with increased thrombin generation, and that thrombin may play a protective role in H. pylori-associated gastroduodenal disorders.     

Benzene increases protein-bound 3-nitrotyrosine in bone marrow of B6C3F1 mice

Benzene, an environmental pollutant, is myelotoxic and leukemogenic in humans. The molecular mechanisms that can account for its biological effects have not been fully elucidated. We hypothesize that one of the underlying mechanism involves nitration of proteins by peroxynitrite and/or by bone marrow myeloperoxidase-dependent pathways in nitric oxide (NO) metabolism. Using 3-nitrotyrosine [Tyr(NO(2))] as a biomarker for NO-induced damage to proteins, we examined the effects of benzene on the levels of Tyr(NO(2)) in bone marrow in vivo. Groups of 8 weeks old B6C3F(1) male mice were given a single i.p. injection of benzene (50, 100, 200 or 400mg/kg bodyweight) in corn oil. The mice in control groups received either no treatment or a single injection of the vehicle. The mice were killed 1h after treatment and proteins were isolated from bone marrow, lung, liver and plasma. The proteins were enzymatically hydrolyzed; amino acids were separated and purified by high pressure liquid chromatography, derivatized, and quantified by electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI-GC/MS). In the GC/MS assay, 3-nitro-l-[(13)C(9)]tyrosine was used as an internal standard and l-[(2)H(4)]tyrosine served to monitor artifactual formation of 3-nitrotyrosine during sample preparation and analysis. We found that treatment of mice with benzene elevates nitration of tyrosine residues in bone marrow proteins. There was a dose (50-200mg benzene/kg b.w.)-dependent increase in protein-bound Tyr(NO(2)) formation (1.5- to 4.5-fold); however, the levels of Tyr(NO(2)) at 400mg benzene/kg b.w. were significantly higher than control but lower than that formed at 200mg benzene/kg b.w. The results of this study, for the first time, indicate that benzene increases protein-bound 3-Tyr(NO(2)) in bone marrow in vivo, and support our previous finding that benzene is metabolized to nitrated products in bone marrow of mice; collectively, these results may in part account for benzene-induced myelotoxicity.     

Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol supports

The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol-disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol-disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12-15-fold comparing optimal derivatives with their just-thiol immobilized counterparts.