Production and properties of inulinase from Aspergillus niger

Summary A thermostable inulinase was identified in a strain of A. niger. The highest activity was observed at 50 °C (50 Lml^–1) and 77% and 34% of this was retained at 60° and 65°C, respectively. pH stability, the effect of thermal stabilizers such as Propylene glycol (10%) and Sorbitol (10%) and effects of different cations were investigated. It was found that the activity was completely inhibited by Ag⁺ and Hg²⁺, while Na⁺ had an activator effect.     

Direct quantification of fungal DNA from soil substrate using real-time PCR

Detection and quantification of genomic DNA from two ecologically different fungi, the plant pathogen Fusarium solani f. sp. phaseoli and the arbuscular mycorrhizal fungus Glomus intraradices, was achieved from soil substrate. Specific primers targeting a 362-bp fragment from the SSU rRNA gene region of G. intraradices and a 562-bp fragment from the F. solani f. sp. phaseoli translation elongation factor 1 alpha gene were used in real-time polymerase chain reaction (PCR) assays conjugated with the fluorescent SYBR(R) Green I dye. Standard curves showed a linear relation (r(2)=0.999) between log values of fungal genomic DNA of each species and real-time PCR threshold cycles and were quantitative over 4-5 orders of magnitude. Real-time PCR assays were applied to in vitro-produced fungal structures and sterile and non-sterile soil substrate seeded with known propagule numbers of either fungi. Detection and genomic DNA quantification was obtained from the different treatments, while no amplicon was detected from non-seeded non-sterile soil samples, confirming the absence of cross-reactivity with the soil microflora DNA. A significant correlation (P<0.0001) was obtained between the amount of genomic DNA of F. solani f. sp. phaseoli or G. intraradices detected and the number of fungal propagules present in seeded soil substrate. The DNA extraction protocol and real-time PCR quantification assay can be performed in less than 2 h and is adaptable to detect and quantify genomic DNA from other soilborne fungi.     

Fullerene‐based inhibitors of HIV‐1 protease

A series of Fmoc‐Phe(4‐aza‐C₆₀)‐OH of fullerene amino acid derived peptides have been prepared by solid phase peptide synthesis, in which the terminal amino acid, Phe(4‐aza‐C₆₀)‐OH, is derived from the dipolar addition to C₆₀ of the Fmoc‐Nα‐protected azido amino acids derived from phenylalanine: Fmoc‐Phe(4‐aza‐C₆₀)‐Lys₃‐OH ( 1 ), Fmoc‐Phe(4‐aza‐C₆₀)‐Pro‐Hyp‐Lys‐OH ( 2 ), and Fmoc‐Phe(4‐aza‐C₆₀)‐Hyp‐Hyp‐Lys‐OH ( 3 ). The inhibition constant of our fullerene aspartic protease PRIs utilized FRET‐based assay to evaluate the enzyme kinetics of HIV‐1 PR at various concentrations of inhibitors. Simulation of the docking of the peptide Fmoc‐Phe‐Pro‐Hyp‐Lys‐OH overestimated the inhibition, while the amino acid PRIs were well estimated. The experimental results show that C₆₀‐based amino acids are a good base structure in the design of protease inhibitors and that their inhibition can be improved upon by the addition of designer peptide sequences. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

HIV persistence: Chemokines and their signalling pathways

Latently infected resting CD4+ T cells are the major barrier to curing HIV. We have recently demonstrated that chemokines, which bind to the chemokine receptors CCR7, CXCR3 and CCR6, facilitate efficient HIV nuclear localisation and integration in resting CD4+ T cells, leading to latency. As latently infected cells are enriched in lymphoid tissues, where chemokines are highly concentrated, this may provide a mechanism for the generation of latently infected cells in vivo. Here we review the role of chemokines in HIV persistence; the main signalling pathways that are involved; and how these pathways may be exploited to develop novel strategies to reduce or eliminate latently infected cells.     

Development and application of a multiplex qPCR technique to detect multiple microcystin-producing cyanobacterial genera in a Canadian freshwater lake

The emergence and persistence of complex blooms comprising multiple toxigenic cyanobacteria genera pose significant challenges for water quality management worldwide. The co-occurrence of morphologically indistinguishable toxic and non-toxic strains makes monitoring and control of these noxious organisms particularly challenging. Conventional monitoring approaches are not only incapable of discriminating toxic from non-toxic strains but also have proven to be less sensitive and specific. In this study, a multiplex quantitative real-time polymerase chain reaction (qPCR) approach was developed and tested for its sensitivity and specificity at detecting, differentiating and estimating potentially toxic Anabaena, Microcystis and Planktothrix genotype compositions in environmental samples. The oligonucleotide primers and probes utilized were designed to target portions of the microcystin synthetase (mcy) E gene that encode synthesis of the unique 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA) moiety of microcystins in the three target genera. Laboratory evaluation showed the developed assay to be highly sensitive and specific at detecting and quantifying targeted genera. Indeed, the assay standards for the Anabaena, Microcystis and Planktothrix reactions attained efficiencies above 90 %, with coefficients of determination consistently above 0.99. Analysis of water samples from Missisquoi Bay, Quebec, Canada, resulted in successful detection and quantification of target toxigenic cyanobacteria even when cell numbers were below the detection limit for the conventional microscopy methods. Furthermore, toxigenic Microcystis spp. were found to be the main putative microcystin-producing cyanobacteria in the study lake. The qPCR technique developed in this study therefore offers simultaneous detection, differentiation and quantification of multiple toxigenic cyanobacteria that otherwise cannot be accomplished by current monitoring approaches.     

The role of the insulin-like growth factors in the central nervous system

Increasing evidence strongly supports a role for insulin-like growth factor-I (IGF-I) in central nervous system (CNS) development. IGF-I, IGF-II, the type IIGF receptor (the cell surface tyrosine kinase receptor that mediates IGF signals), and some IGF binding proteins (IGFBPs; secreted proteins that modulate IGF actions) are expressed in many regions of the CNS beginningin utero. The expression pattern of IGF system proteins during brain growth suggests highly regulated and developmentally timed IGF actions on specific neural cell populations. IGF-I expression is predominantly in neurons and, in many brain regions, peaks in a fashion temporally coincident with periods in development when neuron progenitor proliferation and/or neuritic outgrowth occurs. In contrast, IGF-II expression is confined mainly to cells of mesenchymal and neural crest origin. While expression of type I IGF receptors appears ubiquitous, that of IGFBPs is characterized by regional and developmental specificity, and often occurs coordinately with peaks of IGF expression. In vitro IGF-I has been shown to stimulate the proliferation of neuron progenitors and/or the survival of neurons and oligodendrocytes, and in some cultured neurons, to stimulate function. Transgenic (Tg) mice that overexpress IGF-I in the brain exhibit postnatal brain overgrowth without anatomic abnormality (20–85% increases in weight, depending on the magnitude of expression). In contrast, Tg mice that exhibit ectopic brain expression of IGFBP-1, an inhibitor of IGF action when present in molar excess, manifest postnatal brain growth retardation, and mice with ablated IGF-I gene expression, accomplished by homologous recombination, have brains that are 60% of normal size as adults. Taken together, these in vivo studies indicate that IGF-I can influence the development of most, if not all, brain regions, and suggest that the cerebral cortex and cerebellum are especially sensitive to IGF-I actions. IGF-I’s growth-promoting in vivo actions result from its capacity to increase neuron number, at least in certain populations, and from its potent stimulation of myelination. These IGF-I actions, taken together with its neuroprotective effects following CNS and peripheral nerve injury, suggest that it may be of therapeutic benefit in a wide variety of disorders affecting the nervous system.     

Gas chromatography-mass spectrometry metabolite profiling of worker honey bee (Apis mellifera L.) hemolymph for the study of Nosema ceranae infection

Here, we are presenting a gas chromatography-mass spectrometry (GC/MS) approach for the study of infection of the worker honey bee (Apis mellifera L.) by the newly emerged obligate intracellular parasite Nosema ceranae based on metabolite profiling of hemolymph. Because of the severity of the disease, early detection is crucial for its efficient control. Results revealed that the parasite causes a general disturbance of the physiology of the honey bee affecting the mechanisms controlling the mobilization of energy reserves in infected individuals. The imposed nutritional and energetic stress to the host was depicted mainly in the decreased levels of the majority of carbohydrates and amino acids, including metabolites such as fructose, l-proline, and the cryoprotectants sorbitol and glycerol, which are implicated in various biochemical pathways. Interestingly, the level of glucose was detected at significantly higher levels in infected honey bees. Metabolomics analyses were in agreement with those of multiplex quantitative PCR analyses, indicating that it can be used as a complementary tool for the detection and the study of the physiology of the disease.     

SPIN90/WISH interacts with PSD-95 and regulates dendritic spinogenesis via an N-WASP-independent mechanism

SPIN90/WISH (SH3 protein interacting with Nck, 90 kDa/Wiskott-Aldrich syndrome protein (WASP) interacting SH3 protein) regulates actin polymerization through its interaction with various actin-regulating proteins. It is highly expressed in the brain, but its role in the nervous system is largely unknown. We report that it is expressed in dendritic spines where it associates with PSD-95. Its overexpression increased the number and length of dendritic filopodia/spines via an N-WASP-independent mechanism, and knock down of its expression with small interfering RNA reduced dendritic spine density. The increase in spinogenesis is accompanied by an increase in synaptogenesis in contacting presynaptic neurons. Interestingly, PSD-95-induced dendritic spinogenesis was completely abolished by knock down of SPIN90/WISH. Finally, in response to chemically induced long-term potentiation, SPIN90/WISH associated with PSD-95 and was redistributed to dendritic spines. Our results suggest that SPIN90/WISH associates with PSD-95, and so becomes localized to dendritic spines where it modulates actin dynamics to control dendritic spinogenesis. They also raise the possibility that SPIN90/WISH is a downstream effector of PSD-95-dependent synaptic remodeling.