Molecular cloning,expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica

Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.     

DNA sequencing of 13 cytokine gene fragments of Aotus infulatus and Saimiri sciureus,two non-human primate models for malaria

Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFβ2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most – except IL3 and IL4 – showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus.The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024 and FJ89029.     

A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli proteins

Transformation of engineered Escherichia coli into a robust microbial factory is contingent on precise control of metabolism. Yet, the throughput of omics technologies used to characterize cell components has lagged far behind our ability to engineer novel strains. To expand the utility of quantitative proteomics for metabolic engineering, we validated and optimized targeted proteomics methods for over 400 proteins from more than 20 major pathways in E. coli metabolism. Complementing these methods, we constructed a series of synthetic genes to produce concatenated peptides (QconCAT) for absolute quantification of the proteins and made them available through the Addgene plasmid repository (www.addgene.org). To facilitate high sample throughput, we developed a fast, analytical-flow chromatography method using a 5.5-min gradient (10 min total run time). Overall this toolkit provides an invaluable resource for metabolic engineering by increasing sample throughput, minimizing development time and providing peptide standards for absolute quantification of E. coli proteins.     

Characterization of low-molecular-weight-glutenin subunit genes from the D-genome of Triticum aestivum,Aegilops crassa,Ae. cylindrica and Ae. tauschii

Twenty low-molecular-weight-glutenin subunit (LMW-GS) gene sequences from the D-genome from Aegilops crassa (2n = 4x = 28), Ae. cylindrica (2n = 4x = 28), Ae. tauschii (2n = 2x = 14) and Triticum aestivum (2n = 6x = 42) were obtained using five sets of specific allele primer pairs. Only the sequences of the first primer pair were complete coding sequences (cds) of LMW-GS, and had 305, 304, 306 and 305 LMW-m amino acid residues in Ae. crassa, Ae. cylindrica, Ae. tauschii and T. aestivum, respectively. The repetitive domain and repeat motif numbers of all LMW glutenin subunits showed eight conserved cysteine residues that lead to the same functional activity in different genome. Based on DNA and predicted protein sequences, phylogenetic trees for all sets of sequences were drawn. At the DNA level, the species closest to T. aestivum for the second, third, fourth and fifth set of sequences were Ae. cylindrica, Ae. tauschii and Ae. crassa, respectively. At the protein level, the species closest to T. aestivum based on the first, second and fifth set of sequences were Ae. cylindrica, Ae. crassa and Ae. crassa, respectively. For other sets of sequences, bread wheat proved to be a distinct species. The LMW-GS gene sequences have been recorded in the GenBank with accession numbers JQ726549–JQ726568JQ726549JQ726550JQ726551JQ726552JQ726553JQ726554JQ726555JQ726556JQ726557JQ726558JQ726559JQ726560JQ726561JQ726562JQ726563JQ726564JQ726565JQ726566JQ726567JQ726568.     

Type-IV Antifreeze Proteins are Essential for Epiboly and Convergence in Gastrulation of Zebrafish Embryos

Many organisms in extremely cold environments such as the Antarctic Pole have evolved antifreeze molecules to prevent ice formation. There are four types of antifreeze proteins (AFPs). Type-IV antifreeze proteins (AFP4s) are present also in certain temperate and even tropical fish, which has raised a question as to whether these AFP4s have important functions in addition to antifreeze activity. Here we report the identification and functional analyses of AFP4s in cyprinid fish. Two genes, namely afp4a and afp4b coding for AFP4s, were identified in gibel carp (Carassius auratus gibelio) and zebrafish (Danio rerio). In both species, afp4a and afp4b display a head-to-tail tandem arrangement and share a common 4-exonic gene structure. In zebrafish, both afp4a and afp4b were found to express specifically in the yolk syncytial layer (YSL). Interestingly, afp4a expression continues in YSL and digestive system from early embryos to adults, whereas afp4b expression is restricted to embryogenesis. Importantly, we have shown by using afp4a-specific and afp4b-specifc morpholino knockdown and cell lineage tracing approaches that AFP4a participates in epiboly progression by stabilizing yolk cytoplasmic layer microtubules, and AFP4b is primarily related to convergence movement. Therefore, both AFP4 proteins are essential for gastrulation of zebrafish embryos. Our current results provide first evidence that AFP such as AFP4 has important roles in regulating developmental processes besides its well-known function as antifreeze factors.     

Discovery of novel indole-based aroylhydrazones as anticonvulsants: Pharmacophore-based design

A number of novel melatonin derivatives, containing aroylhydrazone moieties, were synthesized and explored in vivo for anticonvulsant activity, neurotoxicity in ICR mice as well as in-vitro for cytoxicity and oxidative stress in rats. The structures and configurations were confirmed by NMR, FTIR, HRMS and crystal X-ray diffraction method. For selection of potent structures for synthesis a pharmacophore model was used. Two compounds 3e, with a 2-furyl moiety fragment and 3f with 2-thienyl fragment, showed a potency in maximal electroshock (MES) test (ED₅₀ = 50.98 mg kg⁻¹, PI > 5.88 and ED₅₀ = 108.7 mg kg⁻¹; PI > 2.76), respectively, higher than melatonin (ED₅₀ = 160.3 mg kg⁻¹, PI > 1.87). The compounds 3c, 3e, 3f and 3i suppressed psychomotor seizures in the 6 Hz test and 3c was the most potent with higher ED₅₀ = 13.98 mg kg⁻¹ and PI of > 21.46 compared to that of melatonin (ED₅₀ = 49.76 mg kg⁻¹ and PI of > 6.03) in mice. None of the compounds displayed neurotoxicity in the rota-rod test. The novel melatonin derivatives exerted weak cytotoxic effects while 3f showed the lowest hepatoxic effects comparable to that of the positive control melatonin in rats. The high affinities to the elucidated pharmacophore model of the novel melatonin compounds derived from the inclusion of aroylhydrazone moiety in the indole scaffold yielded suitable candidates with anticonvulsant activity in the MES and 6 Hz test of psychomotor seizures.     

Fingerprint scroll wheel launched for mobiles

Designed for integration into laptops, PDA’s and mobile phones, DigitalPersona has announced a space-saving fingerprint sensor/ scroll wheel. The patented U.are.U Firefly roller-style fingerprint reader optically records the fingerprint image as the user glides their finger over the roller.This is a short news story only. Visit www.compseconline.com for the latest computer security industry news.     

Human gut endogenous proteins as a potential source of angiotensin-I-converting enzyme (ACE-I)-, renin inhibitory and antioxidant peptides

It is well known that endogenous bioactive proteins and peptides play a substantial role in the body's first line of immunological defence, immune-regulation and normal body functioning. Further, the peptides derived from the luminal digestion of proteins are also important for body function. For example, within the peptide database BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/en/biopep) 12 endogenous antimicrobial and 64 angiotensin-I-converting enzyme (ACE-I) inhibitory peptides derived from human milk and plasma proteins are listed. The antimicrobial peptide database (http://aps.unmc.edu/AP/main.php) lists over 111 human host-defence peptides. Several endogenous proteins are secreted in the gut and are subject to the same gastrointestinal digestion processes as food proteins derived from the diet. The human gut endogenous proteins (GEP) include mucins, serum albumin, digestive enzymes, hormones, and proteins from sloughed off epithelial cells and gut microbiota, and numerous other secreted proteins. To date, much work has been carried out regarding the health altering effects of food-derived bioactive peptides but little attention has been paid to the possibility that GEP may also be a source of bioactive peptides. In this review, we discuss the potential of GEP to constitute a gut cryptome from which bioactive peptides such as ACE-I inhibitory, renin inhibitory and antioxidant peptides may be derived.     

Interfacial adsorption of antifreeze proteins: a neutron reflection study

Interfacial adsorption from two antifreeze proteins (AFP) from ocean pout (Macrozoarces americanus, type III AFP, AFP III, or maAFP) and spruce budworm (Choristoneura fumiferana, isoform 501, or cfAFP) were studied by neutron reflection. Hydrophilic silicon oxide was used as model substrate to facilitate the solid/liquid interfacial measurement so that the structural features from AFP adsorption can be examined. All adsorbed layers from AFP III could be modeled into uniform layer distribution assuming that the protein molecules were adsorbed with their ice-binding surface in direct contact with the SiO₂ substrate. The layer thickness of 32 Å was consistent with the height of the molecule in its crystalline form. With the concentration decreasing from 2 mg/ml to 0.01 mg/ml, the volume fraction of the protein packed in the monolayer decreased steadily from 0.4 to 0.1, consistent with the concentration-dependent inhibition of ice growth observed over the range. In comparison, insect cfAFP showed stronger adsorption over the same concentration range. Below 0.1 mg/ml, uniform layers were formed. But above 1 mg/ml, the adsorbed layers were characterized by a dense middle layer and two outer diffuse layers, with a total thickness around 100 Å. The structural transition indicated the responsive changes of conformational orientation to increasing surface packing density. As the higher interfacial adsorption of cfAFP was strongly correlated with the greater thermal hysteresis of spruce budworm, our results indicated the important relation between protein adsorption and antifreeze activity.     

Optimization of the cryopreservation of biological resources,Toxoplasma gondii tachyzoites,using flow cytometry

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10⁶ tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me₂SO without incubation. A cooling rate of ∼1 °C/min to −80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.     

Solution Structures,Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.     

Morphology and SSU rDNA-based phylogeny of two Euplotes species from China: E. wuhanensis sp. n. and E. muscicola Kahl, 1932 (Ciliophora,Euplotida)

The living morphology, infraciliature and silverline system of two small Euplotes species, E. wuhanensis sp. n. and E. muscicola Kahl, 1932, isolated from Wuhan, central China, were investigated. Euplotes wuhanensis sp. n. is characterized by a combination of features including small size (40–50 × 25–30 μm), two conspicuously small and eight normal-sized frontoventral cirri, five transverse cirri in two groups, two marginal and two caudal cirri, seven dorsal kineties with about 12 dikinetids in the mid-dorsal row and a double-eurystomus type of dorsal silverline pattern. The Wuhan population of E. muscicola closely resembles previously described populations. The establishments of three subspecies of E. muscicola are not supported. The small subunit ribosomal RNA gene sequences were determined for both species. We propose that the two sequences under the name of E. muscicola (No. AJ305254, DQ917684 deposited in GenBank) are very likely from misidentified material. Phylogenetic analyses based on these data support the validity of both E. muscicola and E. wuhanensis as distinct species.     

Expression profile of rrbp1 genes during embryonic development and in adult tissues of Xenopus laevis

Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19–39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.     

Accumulation of type I fish antifreeze protein in transgenic tobacco is cold-specific

Expression of fish antifreeze protein (AFP) genes in plants is a possible means of increasing their frost resistance and freeze tolerance. Initial work involved transfer into tobacco of an AFP gene from winter flounder which codes for the alanine-rich, -helical Type I AFP. Plants were transformed with a gene construct in which the preproAFP cDNA was inserted between the cauliflower mosaic virus 19S RNA promoter and the nopaline synthetase polyadenylation site. Although transgenic plants produced AFP mRNA, no AFP was detected on western blots. Re-evaluation of AFP expression in these transgenic plants showed that AFP accumulated to detectable levels only after exposure of the plant to cold. Extracts of plants incubated at 4°C for 24 h contained a protein which co-migrated with winter flounder proAFP and was cross-reactive to Type I AFP antisera. Two other minor protein bands of slightly higher apparent M _r also cross-reacted with the antisera and are thought to represent processing intermediates. The proAFP was unique to the transgenic plants and was absent in extracts taken prior to cold exposure. AFP levels increased over the first 48 h of cold incubation then remained stable. Since the -helix content of Type I AFP has been shown to decrease markedly at warmer temperatures, we postulate that Type I AFP stability in transgenic plants is dependent on its secondary structure.     

Drinks containing anthocyanin-rich blackcurrant extract decrease postprandial blood glucose,insulin and incretin concentrations

Blackcurrants are rich in polyphenolic glycosides called anthocyanins, which may inhibit postprandial glycemia. The aim was to determine the dose-dependent effects of blackcurrant extract on postprandial glycemia. Men and postmenopausal women (14 M, 9 W, mean age 46 years, S.D.=14) were enrolled into a randomized, double-blind, crossover trial. Low sugar fruit drinks containing blackcurrant extract providing 150-mg (L-BE), 300-mg (M-BE) and 600-mg (H-BE) total anthocyanins or no blackcurrant extract (CON) were administered immediately before a high-carbohydrate meal. Plasma glucose, insulin and incretins (GIP and GLP-1) were measured 0–120 min, and plasma 8-isoprostane F_2α, together with arterial stiffness by digital volume pulse (DVP) was measured at 0 and 120 min. Early plasma glucose response was significantly reduced following H-BE (n=22), relative to CON, with a mean difference (95% CI) in area over baseline (AOB) 0-30 min of −0.34 mmol/l.h (−0.56, −0.11, P<.005); there were no differences between the intermediate doses and placebo. Plasma insulin concentrations (AOB 0–30 min) were similarly reduced. Plasma GIP concentrations (AOB 0–120 min) were significantly reduced following H-BE, with a mean difference of −46.6 ng/l.h (−66.7, −26.5, P<.0001) compared to CON. Plasma GLP-1 concentrations were reduced following H-BE at 90 min. There were no effects on 8-isoprostane F_2α or vascular function. Consumption of blackcurrant extract in amounts roughly equivalent to 100-g blackcurrants reduced postprandial glycemia, insulinemia and incretin secretion, which suggests that inclusion of blackcurrant polyphenols in foods may provide cardio-metabolic health benefits. This trial was registered at clinicaltrials.gov as NCT01706653.     

黄粉甲抗冻蛋白AFP84a在大肠杆菌中的高效表达及活性检测

为表达和纯化黄粉甲Tenebrio molitor抗冻蛋白, 采用RT-PCR方法扩增得到黄粉甲抗冻蛋白基因afp84a cDNA, 将其连接到pMAL-p2X质粒上, 构建分泌型融合表达载体pMAL-p2X-afp84a, 并在大肠杆菌Escherichia coli TBI中表达; 进一步利用Amylose柱亲和纯化出该重组蛋白, 后利用细菌抗寒性检测重组蛋白的生物活性。结果显示: 融合蛋白含量占总可溶蛋白的40%。SDS-PAGE分析表明, 用MgSO4处理法与超声波细胞破碎法均可使融合蛋白从细胞中释放; 融合蛋白经Amylose柱亲和纯化, Factor Xa因子酶切, 电泳显示获得的目的蛋白呈单一条带。细菌抗寒性检测表明该重组蛋白具有较高的抗冻活性。黄粉甲抗冻蛋白基因afp84a cDNA的克隆、 原核表达为进一步研究抗冻蛋白的性质和应用提供了有用的实验材料。     

Afghan project identifies 150,000 refugees,cuts abuse of system

More than 150,000 Afghan refugees in Pakistan registered their iris patterns as identification during their return journey to Afghanistan over the last year, the UN Refugee Agency has announced.This is a short news story only. Visit www.compseconline.com for the latest computer security news.     

VOICE.TRUST emerges onto European scene

German provider of speaker authentication solutions VOICE.TRUST has formalised its partnership with US company SpeechWorks International. The company, which was launched in 2000, uses SpeechWorks’ SpeechSecure speaker verification technology at the heart of its biometric-based authentication solution VOICE.TRUST Server.This is a short news story only. Visit www.compseconline.com for the latest computer security industry news