UDP-N-ACETYLGLUCOSAMINE-ENOLPYRUVYL TRANSFERASE: DETERMINATION OF PROTONATION STATE OF ACTIVE SITE AMINOACID RESIDUES BY PM6 METHOD. UDP-N-acetylglucosamine-enolpyruvyl transferase (MurA) catalyzes the reaction between phosphoenol pyruvate and UDP-N-acetylglucosamine. We present a theoretical approach using the semiempirical PM6 method for deining protonation state of three active site residues, K22, H125, and K160. Prior comparison with neutron diffraction data showed that PM6 accurately predicted protonation states of active site residues of β-trypsin and D-xylose isomerase. Using the same methodology with MurA crystallographic data, we conclude that when reaction intermediate is located at the active site, H125 and K22 are in protonated form and K160 in neutral form.
There is growing evidence that the sugar N-acetylglucosamine (GlcNAc) plays diverse roles in cell signaling pathways that impact the virulence properties of microbes and host cells. GlcNAc is already well known as a ubiquitous structural component at the cell surface that forms part of bacterial cell wall peptidoglycan, cell wall chitin in fungi and parasites, and extracellular matrix glycosaminoglycans of animal cells. Chitin and peptidoglycan have been previously linked to cell signaling as they can stimulate responses in plant and animal host cells [1–3]. Recent studies now indicate that GlcNAc released from these polymers can also activate cell signaling via several different mechanisms [4–6]. The role of these new GlcNAc signaling path- ways in the regulation of virulence factors will be the focus of this review.
Parkia platycephala lectin 2 was puriﬁed from Parkia platycephala (Legumi- nosae, Mimosoideae) seeds by afﬁnity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the for- mation of three intramolecular disulﬁde bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was speciﬁcally inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydro- lyzed b(1–4) glycosidic bonds linking 2-acetoamido-2-deoxy-b-d-glucopyra- nose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA clo- ning, and its three-dimensional structure, established by X-ray crystallo- graphy at 1.75 A˚ resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (ba) 8 barrel topology harboring the catalytic residues Asp125,
Figure 1 Intersection of the Hexosamine Biosynthetic Pathway (HBP), Phosphoinositide 3-kinase (PI3K)-mTOR-MYC signaling axis, and p53-MDM2 circuit. The HBP (right) generates Uridine diphos- phate N-acetylglucosamine (UDP-GlcNAc) as the end product that is used by the O-GlcNAc transferase (OGT) to covalently attach O-GlcNAc to hydroxyl groups of serine/threonine residues of proteins. This dynamic process is antagonized by O-GlcNAcase (OGA). In cancer, increased HBP flux leads to hyper O-GlcNAcylation. Hyper O-GlcNAcylation of c-Myc activates the phosphoinositide 3-kinase (PI3K)- mTOR-MYC signaling axis (middle). The PI3K pathway cross-talks with Forkhead box M1 (FoxM1), an oncogenic transcription factor that is regulated by levels of O-GlcNAc and OGT (middle). Inflamma- tory responses to genotoxic stress induce activation of NF-κB that can undergo O-GlcNAcylation to me- diate genes in the immune response (left). The loss of p53 activates NF-κB to increase aerobic glycolysis and support tumor metabolism. Hyper O-GlcNAcylation of p53 stabilizes the tumor suppressor and de- creases p53-MDM2 interaction to block proteolysis (bottom). In response to stress, p53 can induce cyclin- dependent kinase inhibitor p21 to inhibit proliferation.
Polysaccharide intercellular adhesin (PIA), also known as poly-N-acetyl-b-(1–6)-glucosamine (PIA/PNAG) is an important component of Staphylococcus aureus biofilms and also contributes to resistance to phagocytosis. The proteins IcaA, IcaD, IcaB, and IcaC are encoded within the intercellular adhesin (ica) operon and synthesize PIA/PNAG. We discovered a mechanism of phase variation in PIA/PNAG expression that appears to involve slipped-strand mispairing. The process is reversible and RecA-independent, and involves the expansion and contraction of a simple tetranucleotide tandem repeat within icaC. Inactivation of IcaC results in a PIA/PNAG-negative phenotype. A PIA/PNAG-hyperproducing strain gained a fitness advantage in vitro following the icaC mutation and loss of PIA/PNAG production. The mutation was also detected in two clinical isolates, suggesting that under certain conditions, loss of PIA/PNAG production may be advantageous during infection. There was also a survival advantage for an icaC-negative strain harboring intact icaADB genes relative to an isogenic icaADBC deletion mutant. Together, these results suggest that inactivation of icaC is a mode of phase variation for PIA/PNAG expression, that high-level production of PIA/PNAG carries a fitness cost, and that icaADB may contribute to bacterial fitness, by an unknown mechanism, in the absence of an intact icaC gene and PIA/PNAG production.
We screened 25,000 microbial extracts consisting of actinomy- cetes and fungi to identify new FabI inhibitors. Meleagrin was isolated from the solid-state fermentation of the fungal strain P. chrysogenum F717. Meleagrin was previously isolated from P. meleagrinum  and P. chrysogenum , but its biological activity, including antimicrobial activity, has not been reported. Although its activity was weak, meleagrin clearly showed inhibition selective for S. aureus FabI over S. pneumoniae FabK. Importantly, the binding of meleagrin with S. aureus FabI was demonstrated by the fluorescence quenching assay. Furthermore, its inhibition of FabI was supported by results obtained using its chemical derivatives, the intracellular fatty acid synthesis assay, and the fabI-overex- pressing assay. Interestingly, meleagrin and its more active derivatives showed antibacterial activity against S. pneumoniae, in which FabK is the sole enoyl-ACP reductase, and it did not produce spontaneously resistant mutants of S. aureus or E. coli, in contrast to triclosan, which suggests that meleagrin inhibits multiple targets. Meleagrin inhibited the incorporation of radio- labeled acetate into lipids in S. pneumoniae and S. aureus, whereas incorporation of thymidine (DNA), uridine (RNA), isoleucine (protein), and N-acetylglucosamine (cell wall) was not inhibited, which indicates that these compounds inhibit fatty acid synthesis through one or more modes of action in addition to FabI inhibition. The multitarget effect was confirmed by the fabK- overexpression assay in E. coli. The multitarget effect is very important from the point of view of drug development because a single point mutation in one gene for a drug with a single target renders the strain resistant and the drug useless. Thus, when considering that one of the advantages of antibacterial agents having multiple targets is the reduced development of drug resistance , meleagrin and its derivatives hold promise for the development of new antibiotics that can treat infections caused by multidrug-resistant pathogens.
The presence of an additional sulphur atom on the diamide struc- tures originating thiodiglycolamide derivatives has a positive eﬀect on Pd(II) extraction, as DMDCHTDGA is able to extract Pd(II) from HCl solutions independently of their concentration (from 1 M to 8 M), which is in accordance with the behavior previously described for TOTDGA, despite the use of a diﬀerent diluent (a mixture of 80% n-dodecane and 20% 2-ethylhexanol). A similar Pd(II) extraction pro ﬁle is also pre- sented by DMDPHTDGA in chloroform, proving that, in this case, the replacement of the cyclohexyls by the phenyl substituents does not a ﬀect the metal extraction. Contrarily, the replacement by butyl groups resulting in DMDBTDGA appears to have had an adverse e ﬀect, since Pd (II) is only quantitatively extracted from 1.5 M to 4.5 M HCl solutions, decreasing gradually for higher acid concentrations, although the use of a di ﬀerent diluent should also be considered.
Em balão de fundo redondo de 50 mL foram solubilizados 0,28 mmol 28 ou 29 e 0,84 mmol de cloreto de sulfonila correspondente (RSO 2 Cl) em 3 mL de THF anidro. A mistura foi mantida sob agitação magnética a 0 °C (banho de gelo). A cada 30 minutos foi adicionada uma solução de 0,1 mmol de Et 3 N em 0,5 mL de THF, totalizando 0,4 mmol de Et 3 N no intervalo de 2 horas. O material foi mantido sob
Na região de baixa freqüência, surgiu uma nova banda em 407 cm -1 que foi atribuída a ligação Sn-N [2,3,4]. Outro fato que deve ser destacado é a ausência de uma banda relativa à ligação Sn-Cl, presente no material de partida em 335 cm -1 . Isso indica que o átomo de cloro saiu da esfera de coordenação do átomo de estanho, podendo estar como contra-íon. Uma provável estrutura para este composto, que estaria de acordo com os dados da espectroscopia no infravermelho e com a análise elementar é apresentada na Figura 11.
A Prefeitura Municipal de Cachoeira da Prata/MG, realizará procedimento de licitação nº 018/2017, modalidade, Pregão Presencial para Registro de Preços, tipo menor preço, nos termos da Lei 10.520/02, e legislação correlata; em especial a Lei federal 8.666/93, e de acordo com as condições estabelecidas no presente instrumento convocatório e seus anexos. Os envelopes contendo a proposta comercial e documentação de habilitação serão recebidos em sessão pública às 09:00 horas do dia 06/04/2017, na sala da Comissão Permanente de Licitação da Prefeitura Municipal, situada na Praça JK, n° 139, Centro, Cidade de Cachoeira da Prata/MG, tel.: (31)3716-1392 - oportunidade em que serão examinados. O pregão será realizado pelo Pregoeiro oficial, ou substituto designados pela portaria nº 013/2017 de 02 de janeiro de 2017.
A resposta ´e d = 31 lanc¸amentos. A ideia ´e pen- sar que o n ´umero em cada face representa uma casa (6 n ´umeros = 6 casas). Queremos alguma casa com mais do que d pombos (lanc¸amentos) ent˜ao deve-se distribuir os resultados dos lanc¸amentos nas respectivas casas. Se tivermos 6d + 1 lanc¸amentos, n˜ao ´e poss´ıvel que cada n ´umero tenha sa´ıdo no m´aximo d vezes e assim teremos uma casa com pelo menos d + 1 pombos.
moment of structure 1) is recovered. An increase of 0.8 D (that is, from 4.282 to 5.082 D, see Table 1) in the dipole moment can cause an increase of about 57 kJ/mol (*13.6 kcal/mol) in the ion–dipole interaction [ 22], if one uses the distance of about 3.47 A ˚ between the Fe(III) and the center of mass of the N-nitrosodimethylamine . Such increase may play an important role in the stabilization of the Fe(III)–nitrosamine interaction, assuming that it has a substantial electrostatic contribution, although repulsive interaction (e.g. Pauli repulsion ) should also be of significant strength, otherwise the Fe(III)–nitrosamine interaction would not be weak , since the value of the ion–dipole interaction energy is *309 kJ/mol for a dipole moment of 4.282 D (see Table 1) at a distance of 3.47 A ˚ from the Fe(III) ion. Structure 14 (see Table 1; Fig. 2) has a dipole moment very close to, though slightly smaller than, the dipole moment of structure 1 and can thus result in practically the same strength of the ion–dipole interac- tion. The largest deviations from planarity are obtained for this latter structure, with values of 4.8° and 7.9° for the \C1NNO dihedral angle and 7.8° and 12.0° for the \C2NNO dihedral angle, at the B3LYP and MP2 levels, respectively (see Table 1). It is important to point out that the dipole moment of the coordinated N-nitrosodimethyl- amine is significantly higher (by about 0.7–0.8 D), at both computational levels, than the dipole moment of the free molecule (see Table 1). On the other hand its polarizability is significantly lower (by about 4.0 bohr 3 ) than the polar- izability of the free molecule (see Table 1), also at both computational levels. However, either using the geometry of the free or coordinated molecule the attractive ion– induced dipole interaction is expected to be of minor importance as compared to the ion–dipole interaction, if one takes into account the aforementioned values of the dipole moment and ion–dipole distance.
Exerc´ıcio 18. Suponha que dispomos de uma caixa bem grande em que ´e realizado o experimento descrito a se- guir. No primeiro minuto uma bola ´e colocada na caixa, no segundo minuto trˆes bolas s˜ao acrescentadas, no terceiro minuto outras cinco bolas e assim, sucessivamente, a cada minuto acrescenta-se `a caixa a pr ´oxima quantidade ´ımpar de bolas subsequente `a quantidade acrescentada no minuto anterior. Em outras palavras, no minuto n s˜ao acrescentadas 2n − 1 bolas.
Upon reaching adult hood, it was observed that the average longevity was 13.3 days (Table 2). The adults measure 9.1 mm long by 4.1 mm in chest width (n = 10). They have light brown thorax and elytra. The head is dark tending to black in the frontal region between the eyes, and on the clypeus the color is dark brown. The biological cycle was completed with an average of 211.2 days, which allows for the formation of one generation per year, characterizing the species as univoltine.