Laccase-catalysed synthesis of coupling products of phenolic substrates in different reactors

Substrate oxidation of aromatic substances by the enzyme laccase followed by a heteromolecular coupling with a co-substrate is a promising possibility for the synthesis of new compounds. To find a suitable reactor for the effective production of new compounds, the laccase-catalysed coupling of 3-(3,4-dihydroxyphenyl)propionic acid with 4-aminobenzoic acid was investigated as a model system. Based on the kinetic parameters, a mathematical model was used to predict the reaction yield and oxygen demand in a discontinuously stirred tank reactor and a continuously operated stirred tank reactor. Membrane processes were used for bubble-free aeration of the system and to recover the soluble enzyme.     

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

Background

CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively.

Results

Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure.

Conclusion

Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.     

Modelling the reaction course of N-acetylneuraminic acid synthesis from N-acetyl-d-glucosamine—new strategies for the optimisation of neuraminic acid synthesis

In this work, a model describing the complete enzyme catalysed synthesis of N-acetylneuraminic acid (Neu5Ac) from N-acetyl-d-glucosamine (GlcNAc) is presented. It includes the combined reaction steps of epimerisation from GlcNAc to N-acetyl-d-mannosamine (ManNAc) and the aldol condensation of ManNAc with sodium pyruvate yielding Neu5Ac. The model is expedient to predict the reaction course for various initial and feed concentrations and therefore to calculate reaction times and yields. The equilibrium constants calculated from the kinetic constants via the Haldane relationship correspond with experimental values very well (0.26 calculated and 0.24 experimental value for the epimerisation, 27.4 l mol⁻¹ calculated and 28.7 l mol⁻¹ experimental for the aldol condensation). The actual relevance of the model is shown by a scale-up. Using the model, an optimisation of reaction conditions in consideration of different targets is possible. Exemplarily, it is presented how the optimal ratio of the two enzymes in the reaction can be determined and how the composition of the reaction solution in a fed-batch reactor can be designed to meet downstream processing needs.     

Membrane TNF confers protection to acute mycobacterial infection

Background

Tumour necrosis factor (TNF) is crucial for the control of mycobacterial infection as TNF deficient (KO) die rapidly of uncontrolled infection with necrotic pneumonia. Here we investigated the role of membrane TNF for host resistance in knock-in mice with a non-cleavable and regulated allele (mem-TNF).

Methods

C57BL/6, TNF KO and mem-TNF mice were infected with M. tuberculosis H37Rv (Mtb at 100 CFU by intranasal administration) and the survival, bacterial load, lung pathology and immunological parameters were investigated. Bone marrow and lymphocytes transfers were used to test the role of membrane TNF to confer resistance to TNF KO mice.

Results

While TNF-KO mice succumbed to infection within 4–5 weeks, mem-TNF mice recruited normally T cells and macrophages, developed mature granuloma in the lung and controlled acute Mtb infection. However, during the chronic phase of infection mem-TNF mice succumbed to disseminated infection with necrotic pneumonia at about 150 days. Reconstitution of irradiated TNF-KO mice with mem-TNF derived bone marrow cells, but not with lymphocytes, conferred host resistance to Mtb infection in TNF-KO mice.

Conclusion

Membrane expressed TNF is sufficient to allow cell-cell signalling and control of acute Mtb infection. Bone marrow cells, but not lymphocytes from mem-TNF mice confer resistance to infection in TNF-KO mice. Long-term infection control with chronic inflammation likely disrupting TNF mediated cell-cell signalling, additionally requires soluble TNF.     

Diel movement of smallmouth yellowfish Labeobarbus aeneus in the Vaal River,South Africa

Diel movements of Orange–Vaal smallmouth yellowfish Labeobarbus aeneus (Burchell, 1822) in the Vaal River, South Africa, were determined by externally attaching radio transmitters to 11 adult fish and manually tracking them between March and May 2012. Twenty-four radio telemetry monitoring surveys produced 2 304 diel tracks. At night, yellowfish displayed a preference for slow shallow (<0.3?m s?1, <0.5?m) and fast shallow habitats (>0.3?m s?1, <0.3?m), whereas by day they avoided these habitats, preferring fast deep areas (>0.3?m s?1, >0.3?m). The average total distance of 272?m moved per 24-hour period was three times greater than the diel range, and the average maximum displacement per minute was significantly higher in daytime (4?m) than at night (1.5?m). These findings suggest that L. aeneus is active primarily during the day in fast-flowing, deeper waters, and relatively inactive at night, when it occupies shallower habitats. This behaviour should be further explored to identify causal mechanisms underlying the diel habitat shifts in this species such as water temperature, foraging tactics and/or predator avoidance.     

Defensive insect symbiont leads to cascading extinctions and community collapse

Animals often engage in mutualistic associations with microorganisms that protect them from predation, parasitism or pathogen infection. Studies of these interactions in insects have mostly focussed on the direct effects of symbiont infection on natural enemies without studying community‐wide effects. Here, we explore the effect of a defensive symbiont on population dynamics and species extinctions in an experimental community composed of three aphid species and their associated specialist parasitoids. We found that introducing a bacterial symbiont with a protective (but not a non‐protective) phenotype into one aphid species led to it being able to escape from its natural enemy and increase in density. This changed the relative density of the three aphid species which resulted in the extinction of the two other parasitoid species. Our results show that defensive symbionts can cause extinction cascades in experimental communities and so may play a significant role in the stability of consumer‐herbivore communities in the field.     

Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One- and Two-Phase Systems

This review discusses recent achievements in the field of cofactor regeneration for the nicotinamide cofactors NADH and NADPH. The examples discussed include alcohol dehydrogenases, formate dehydrogenase, glucose dehydrogenase and a hydrogenase. For the reaction either one-phase systems or two-phase systems in combination with an organic solvent are discussed. For the enantioselective reduction of 2-octanone to (R)-2-octanol it could be shown that enzyme coupled NADPH regeneration with glucose dehydrogenase and glucose results in shorter reaction times and higher yields when compared to the substrate coupled regeneration with 2-propanol.     

Distinct roles for intra- and extracellular siderophores during Aspergillus fumigatus infection

Siderophore biosynthesis by the highly lethal mould Aspergillus fumigatus is essential for virulence, but non-existent in humans, presenting a rare opportunity to strategize therapeutically against this pathogen. We have previously demonstrated that A. fumigatus excretes fusarinine C and triacetylfusarinine C to capture extracellular iron, and uses ferricrocin for hyphal iron storage. Here, we delineate pathways of intra- and extracellular siderophore biosynthesis and show that A. fumigatus synthesizes a developmentally regulated fourth siderophore, termed hydroxyferricrocin, employed for conidial iron storage. By inactivation of the nonribosomal peptide synthetase SidC, we demonstrate that the intracellular siderophores are required for germ tube formation, asexual sporulation, resistance to oxidative stress, catalase A activity, and virulence. Restoration of the conidial hydroxyferricrocin content partially rescues the virulence of the apathogenic siderophore null mutant Delta sidA, demonstrating an important role for the conidial siderophore during initiation of infection. Abrogation of extracellular siderophore biosynthesis following inactivation of the acyl transferase SidF or the nonribosomal peptide synthetase SidD leads to complete dependence upon reductive iron assimilation for growth under iron-limiting conditions, partial sensitivity to oxidative stress, and significantly reduced virulence, despite normal germ tube formation. Our findings reveal distinct cellular and disease-related roles for intra- and extracellular siderophores during mammalian Aspergillus infection.