Crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A(2) complexed with p-bromophenacyl bromide and alpha-tocopherol inhibitors at 1.9-and 1.45-A resolution

An acidic phospholipase A(2) (PLA(2)) isolated from Bothrops jararacussu snake venom was crystallized with two inhibitors: alpha-tocopherol (vitamin E) and p-bromophenacyl bromide (BPB). The crystals diffracted at 1.45- and 1.85-Angstrom resolution, respectively, for the complexes with alpha-tocopherol and p-bromophenacyl bromide. The crystals are not isomorphous with those of the native protein, suggesting the inhibitors binding was successful and changes in the quaternary structure may have occurred. (C) 2004 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction studies of two myotoxic Lys49-phospholipases A(2) complexed with alpha-tocopherol

BnSP-7 and BnSP-6, two Lys49-phospholipase A(2) isolated from Bothrops neuwiedi pauloensis snake venom, were co-crystallized with a-tocopherol and X-ray diffraction data were collected for both complexes (2.2 and 2.6 angstrom). A new alternative quaternary conformation for these two complexes compared with all other dimeric Lys49-PLA(2) has been observed.

A stability transition at mildly acidic pH in the alpha-hemolysin (alpha-toxin) from Staphylococcus aureus

The effects of mildly acidic conditions on the free energy of unfolding (Delta G(u)(buff)) of the pore-forming alpha-hemolysin (alpha HL) from Staphylococcus aureus were assessed between pH 5.0 and 7.5 by measuring intrinsic tryptophan fluorescence, circular dichroism and elution time in size exclusion chromatography during urea denaturation, Decreasing the pH from 7.0 to 5.0 reduced the calculated Delta G(u)(buff) from 8.9 to 4.2 kcal moI(-1), which correlates with an increased rate of pore formation previously observed over the same pH range, It is proposed that the lowered surface pH of biological membranes reduces the stability of alpha HL thereby modulating the rate of pore formation. (C) 1999 Federation of European Biochemical Societies.

Stochastic variational approach applied to 2, 3 and 4 alpha particles

A stochastic variational method is applied to calculate the binding energies and root-mean-square radii of 2, 3 and 4 alpha particles using an S-wave Ali-Bodmer potential. The results agree with other calculations. We discuss the application of the present method to study the universality in weakly-bound three and four-body systems in the context of ultracold atomic traps.

The N terminus of the human alpha(1D)-adrenergic receptor prevents cell surface expression

We previously reported that truncation of the N-terminal 79 amino acids of alpha(1D)-adrenoceptors (Delta(1-79)alpha(1D)-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in alpha(1D)-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length alpha(1D)-ARs were found primarily in intracellular compartments, whereas Delta(1-79)alpha(1D)-ARs were translocated to the plasma membrane. This resulted in a 3- to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of alpha(1)-ARs containing the body of one subtype and the N terminus of another (alpha(1A) NT-D, alpha(1B) NT-D, alpha(1D) NT-A, and alpha(1D)NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of alpha(1A)- or alpha(1B)-ARs containing the alpha(1D)-N terminus decreased by 86 to 93%, whereas substitution of alpha(1A)- or alpha(1B)-N termini increased alpha(1D)-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged alpha(1D)NT-B-ARs were found only on the cell surface, whereas GFP-tagged alpha(1B)NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged alpha(1D)- and Delta(1-79)alpha(1D)-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express alpha(1D)-ARs. These findings demonstrate that the N-terminal region of alpha(1D)-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.

REGULATION OF BOVINE ENDOMETRIAL SECRETION OF PROSTAGLANDINS AND SYNTHESIS OF 2',5'-OLIGOADENYLATE SYNTHETASE BY INTERFERON-ALPHA MOLECULES

Experiments were performed to (1) verify the inhibitory effect of bovine trophoblast protein-1 (bTP-1) on uterine prostaglandin synthesis, (2) evaluate whether other interferon-alpha (IFN-alpha) molecules also inhibit prostaglandin secretion, and (3) test whether the enzyme 2',5'-oligoadenylate synthetase (2-5A synthetase) can be induced in endometrium by interferon-alpha. In experiment 1, all interferon molecules (bTP-1, oTP-1, bIFN-alpha and hIFN-alpha) equally inhibited secretion of PGF and PGE2 from endometrial explant cultures obtained at day 17 of the estrous cycle. In experiment 2, endometrial explants obtained from day 17 of the cycle were cultured with and without bovine serum albumin (BSA; 50-mu-g/ml) and bIFN-alpha (0, 0.84, 4.2, and 42 nM). Addition of BSA to the culture medium greatly enhanced the accumulation of PGF into the medium. The bIFN-alpha inhibited accumulation of PGF and PGE2 in both the presence or absence of BSA by 12 h. All three concentrations of bIFN-alpha were equally effective in inhibiting prostaglandin accumulation. Additionally, all concentrations of bIFN-alpha increased the amounts of 2-5A synthetase in endometrium. In conclusion, these results confirm the inhibitory effect of bTP-1 on PGF release from endometrium and demonstrate that bTP-1 can also inhibit PGE2 secretion. Furthermore, other interferon-alpha molecules, including bIFN-alpha, hIFN-alpha, and oTP-1, also reduced PGF and PGE2 secretion in culture. It is likely, therefore, that conceptus and other interferon-alpha molecules exert similar effects on endometrium in vitro and that the antiluteolytic effects of bIFN-alpha in vivo are mediated in part by changes in endometrial prostaglandin synthesis.

13,14-Dihydro-15-Keto Prostaglandin F-2 alpha Release in Response to Oxytocin Challenge Early Post-Partum in Anoestrous Nelore Cows Submitted to Temporary Calf Removal and Progesterone Priming

The study evaluated, in early post-partum anoestrous Nelore cows, if the increase in plasma oestradiol (E2) concentrations in the pre-ovulatory period and/or progesterone priming (P4 priming) preceding ovulation, induced by hormonal treatment, reduces the endogenous release of prostaglandin PGF(2)alpha and prevents premature lysis of the corpus luteum (CL). Nelore cows were subjected to temporary calf removal for 48 h and divided into two groups: GPE/eCG group (n = 10) and GPG/eCG group (n = 10). Animals of the GPE/eCG group were treated with a GnRH agonist. Seven days later, they received 400 ID of eCG, immediately after PGF(2)alpha treatment, and on day 0, 1.0 mg of oestradiol benzoate (EB). Cows of the GPG/eCG group were similarly treated as those of the GPE/eCG group, except that EB was replaced with a second dose of GnRH. All animals were challenged with oxytocin (OT) 9, 12, 15 and 18 days after EB or GnRH administration and blood samples were collected before and 30 min after OT. Irrespective of the treatments, a decline in P4 concentration on day 18 was observed for cows without P4 priming. However, animals exposed to P4 priming, treated with EB maintained high P4 concentrations (8.8 +/- 1.2 ng/ml), whereas there was a decline in P4 on day 18 (2.1 +/- 1.0 ng/ml) for cows that received GnRH to induce ovulation (p < 0.01). Production of 13,14-dihydro-15-keto prostaglandin F-2 alpha (PGFM) in response to OT increased between days 9 and 18 (p < 0.01), and this increase tended to be more evident in animals not exposed to P4 priming (p < 0.06). In conclusion, the increase in E2 during the pre-ovulatory period was not effective in inhibiting PGFM release, which was lower in P4-primed than in non-primed animals. Treatment with EB promoted the maintenance of elevated P4 concentrations 18 days after ovulation in P4-primed animals, indicating a possible beneficial effect of hormone protocols containing EB in animals with P4 priming.

Conserved sequences in the β subunit of archaeal and eukaryal translation initiation factor 2 (elF2), absent from elF5, mediate interaction with elF2γ

The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode. This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5' end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome. Here we provide a comparative sequence analysis of the β subunit of eIF2 and its archaeal counterpart (aIF2β). aIF2β differs from eIF2β in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B. The remaining sequences are highly conserved, and are shared with eIF5. Previously isolated mutations in the yeast eIF2β, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2β, but not in eIF5. We show that the sequence of eIF2B homologous to aIF2β is sufficient for binding eIF2γ, the only subunit with which it interacts, and comprises, at the most, 78 residues, eIF5 does not interact with eIF2γ, despite its similarity with eIF2β, probably because of a gap in homology in this region. These observations have implications for the evolution of the mechanism of translation initiation.

Two short peptides including segments of subunit A of Escherichia coli DNA gyrase as potential probes to evaluate the antibacterial activity of quinolones

Quinolones constitute a family of compounds with a potent antibiotic activity. The enzyme DNA gyrase, responsible for the replication and transcription processes in DNA of bacteria, is involved in the mechanism of action of these drugs. In this sense, it is believed that quinolones stabilize the so-called 'cleavable complex' formed by DNA and gyrase, but the whole process is still far from being understood at the molecular level. This information is crucial in order to design new biological active products. As an approach to the problem, we have designed and synthesized low molecular weight peptide mimics of DNA gyrase. These peptides correspond to sequences of the subunit A of the enzyme from Escherichia coli, that include the quinolone resistance-determining region (positions 75-92) and a segment containing the catalytic Tyr-122 (positions 116-130). The peptide mimic of the non-mutated enzyme binds to ciprofloxin (CFX) only when DNA and Mg2+ were present (Kd = 1.6 × 10 -6 m), a result previously found with DNA gyrase. On the other hand, binding was reduced when mutations of Ser-83 to Leu-83 and Asp-87 to Asn-87 were introduced, a double change previously found in the subunit A of DNA gyrase from several CFX-resistant clinical isolates of E. coli. These results suggest that synthetic peptides designed in a similar way to that described here can be used as mimics of gyrases (topoisomerases) in order to study the binding of the quinolone to the enzyme-DNA complex as well as the mechanism of action of these antibiotics. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.

Aplicação do Método de Rietveld em caracterização estrutural e nanoestrutural do espinélio 'Ni IND. 1'- 'delta' Co'delta''Fe IND.2''O IND.4' preparado por reação de combustão

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mobilidade de oxigênio intersticial em SM'BA IND.2' C IND.U 'IND.3' 'O IND. 7-'delta' medida através de espectroscopia mecânica

Pós-graduação em Ciência e Tecnologia de Materiais - FC

Ferritas 'MG IND. 1' - 'delta'ZN''delta''FE' IND. 2'O IND. 4' obtidas por síntese de reação de combustão: caracterização estrutural e nanoestrutural

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)