Prediction, isolation, overexpression and antifungal activity analysis of Medicago truncatula var. truncatula putative thaumatin like proteins (TLP-1, -2, -3, -4 and -5).

Pathogenesis-related proteins (PR-proteins) are induced in response to environmental stresses such as osmotic and drought stress, wounding, microbial infections and treatment with specific plant hormones and elicitors. These proteins are classified into several groups (PR-1 through PR-17) based on their amino acid sequence and biochemical functions. The present study focuses on prediction, isolation, over-expression and analysis of the antifungal activities of the thaumatin-like proteins (i.e. PR-5) in the model legume M. truncatula var. truncatula. Analysis of M. truncatula genome sequence, available freely on the NCBI website, indicated the presence of at least 15 PR-5 Open Reading Frames (ORFs), 5 of them (dubbed TLP-1, -2, -3, -4 and -5) were selected for this study. Using gene-specific primers, the genomic coding sequences were isolated, sequenced and all confirmed to match with those reported in the database. All the fragments were, then, cloned in Escherichia coli isolate BL21 (DE3), using pET-21c(+) plasmids for subsequent overexpression (overexpression). All 5 genes were expressed as inclusion bodies (IBs) with masses, estimated by SDS PAGE, corresponding to the theoretical values. As expected, none of the protein IBs had no detectable effect on the phytopathogenic fungi Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium solani, Verticillium sp. and Phytophtora spp. However, when the in vitro refolded IB preparations were applied, all displayed comparable strong antifungal activities against the tested fungi. The current study is the first report of overexpression and evaluation of antifungal activities of PR-5 family of proteins from M. truncatula Var. truncatula, and provides experimental evidence that all investigated proteins have the potential for enhancing resistance against some important fungal pathogens.

Grade distribution digests: A novel tool to enhance teaching and student learning in laboratory practicals.

Assessment is a central component of course curriculums and is used to certify student learning, but it can also be used as a tool to improve teaching and learning. Many laboratory courses are structured such that there is only a grade for a particular laboratory, which limits the insights that can be gained in student learning. We developed a laboratory program that incorporates assessments designed to probe student understanding of different components of the individual modules making up the program. The challenge was to analyze and present grades from these assessment tasks in a format that was readily interpretable by academics. We show that a simplified synthesis of grade distributions (grade distribution digests) provides sufficient information to make decisions about changes in course components. The main feature of the digests is its data visualization approach, where student grades for individual laboratory practicals, individual assessment tasks or individual assessment items are graphically presented as an overall average grade, an average top quartile grade and an average bottom quartile grade, and relative averages across all assessments. This ability to visualize student grades in variety of contexts enables academics with many other demands on their time (e.g. research and administration) to more efficiently identify ways to improve teaching delivery and learning outcomes. Examples are presented of the use of such data to identify and improve deficiencies in both student skills and teaching practice, resulting in improved learning outcomes. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):130-140, 2018.

Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ.

AIM: To identify and characterize the effect of phosphorylation on the subcellular localization of Ankrd54. METHODS: HEK293T cells were treated with calyculin A, staurosporin or phorbol 12-myristate 13-acetate (PMA). Cells were transfected with eGFP-tagged Ankrd54 with or without Lyn tyrosine kinase (wild-type, Y397F mutant, or Y508F mutant). The subcellular localization was assessed by immunofluorescence imaging of cells, immunoblotting of subcellular fractionations. The phosphorylation of Ankrd54 was monitored using Phos-tagTM gel retardation. Phosphorylated peptides were analysed by multiple-reaction-monitoring (MRM) proteomic analysis. RESULTS: Activation of PKC kinases using PMA promoted nuclear export of Ankrd54 and correlated with increased Ankrd54 phosphorylation, assayed using Phos-tagTM gel retardation. Co-expression of an active form of the PKCδ isoform specifically promoted both phosphorylation and cytoplasmic localization of Ankrd54, while PKCδ, Akt and PKA did not. Alanine mutation of several serine residues in the amino-terminal region of Ankrd54 (Ser14, Ser17, Ser18, Ser19) reduced both PMA induced cytoplasmic localization and phosphorylation of Ankrd54. Using MRM proteomic analysis, phosphorylation of the Ser18 residue of Ankrd54 was readily detectable in response to PMA stimulation. PMA stimulation of cells co-expressing Ankrd54 and Lyn tyrosine kinase displayed increased co-immunoprecipitation and enhanced co-localization in the cytoplasm. CONCLUSION: We identify phosphorylation by PKCδ as a major regulator of nuclear-cytoplasmic shuttling of Ankrd54, and its interaction with the tyrosine kinase Lyn.

Recombinant production, purification and characterization of vessel dilator in E. coli.

Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E. coli BL21 (DE3), was recorded with VDII that carried the shortest fusion partner. Subsequent to the initial capture of the fusion protein by a Ni affinity column, the vessel dilator monomers were cleaved by trypsin treatment, and further purified to at least 90% homogeneity by anion exchange chromatography. De-novo sequencing and in vivo anticancer activity tests were used to verify the peptide sequence and its biological activity, respectively. The final yield was estimated to be approximately 15 mg of the purified vessel dilator per gram wet weight of the bacterial cells.

Long-term survival of high quality sperm: insights into the sperm proteome of the honeybee Apis mellifera.

In the social bees, ants, and wasps, females (queens) mate only during a brief period early in their lives and afterward store a lifetime supply of sperm in a specialized organ, the spermatheca. In some species, stored sperm can remain viable for several decades and is used by queens to fertilize millions of eggs. The physiological adaptations that allow this prolonged survival are unknown. To unravel them, we conducted proteomic analyses on the sperm of the honeybee Apis mellifera to define proteins that are bee-specific or highly divergent from sequences in the sperm proteomes of flies or mammals and might therefore be associated with long-term sperm survival. We identified a honeybee sperm proteome of 336 members and defined the subset of proteins or protein networks that cannot be discerned in the sperm proteomes of fruit flies and humans. This subset contained a significant number of proteins that are predicted to act in enzyme regulation or in nucleic acid binding and processing. From our analysis we conclude that long-term survival of sperm in social insects could be underpinned by substantial changes in only a specific subset of sperm proteins that allow physiological adaptation to storage. The unexpected preponderance of proteins predicted to be involved in transcriptional processes and enzyme regulation suggest these are the primary targets of this adaptation.

Seminal fluid proteins differ in abundance between genetic lineages of honeybees.

Seminal fluid is transferred to the females' reproductive tract as part of the ejaculate and contains highly complex molecular machinery that is of central importance for male and female reproductive success. Interspecific studies suggest rapid evolutionary changes in the sequences of some seminal fluid proteins and also highlight the importance of specific seminal fluid proteins for sperm function and paternity success. Much less work has been conducted to study if variation in the steady-state abundance of seminal fluid proteins occurs within a species, which could provide a foundation for future selection to act upon. Here we used a unique breeding program of the honeybee Apis mellifera to provide evidence for quantified differences in seminal fluid protein abundances between three genetic lineages that have been bred for ~20 generations. We found the same subset of seminal fluid proteins to be present in all lines, but protein abundance or protein modification state varied significantly for 16% of the protein spots investigated. Protein spots with changed abundances were identified using mass spectrometry, with the abundance of a number documented from other species to be correlated with male fertility, reproductive success or immune-competence. We conclude that significant alterations in the abundance or modification state of specific proteins in seminal fluid can be linked to different genotypes in honeybees.

A serine proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph.

Activation of prophenoloxidase (proPO) in insects is a defense mechanism against intruding microorganisms and parasites. Pattern recognition molecules induce activation of an enzymatic cascade involving serine proteinases, which leads to the conversion of proPO to active phenoloxidase (PO). Phenolic compounds produced by pPO-activation are toxic to invaders. Here, we describe the isolation of a venom protein from the parasitoid, Cotesia rubecula, injected into the host, Pieris rapae, which is homologous to serine proteinase homologs (SPH). The data presented here indicate that the protein interferes with the proteolytic cascade, which under normal circumstances leads to the activation of proPO and melanin formation.

Isolation and characterization of a novel venom protein from an endoparasitoid, Cotesia rubecula (Hym: Braconidae).

Insects are important vectors of diseases with remarkable immune defense capabilities. Hymenopteran endoparasitoids are adapted to overcome the host defense system and, therefore, are useful sources of immune-suppressing proteins. Not much is known about venom proteins in endoparasitoids, especially those that have a functional relationship with polydnaviruses (PDVs). Here, we describe the isolation and characterization of a small venom protein (Vn4.6) from an endoparasitoid, Cotesia rubecula, which interferes with the activation of the host hemolymph prophenoloxidase. The coding region for Vn4.6 is located upstream in the opposite direction of a gene coding for a C. rubecula PDV-protein (Crp32).

Isolation of fungal cell wall degrading proteins from barley (Hordeum vulgare L.) leaves infected with Rhynchosporium secalis.

Proteins with antifungal activity towards Rhynchosporium secalis conidia were isolated from the intercellular washing fluid (IWF) of barley leaves. The active components were purified by high-performance liquid chromatography under conditions that maintained biological activity. Five major barley IWF proteins deleterious to the cell wall of viable R. secalis conidia were isolated and identified by a combination of N-terminal amino acid sequencing, peptide mapping, and determination of mass and isoelectric point. They were a 32-kDa beta-1,3-glucanase (Pr32), a 25-kDa chitinase (Pr25), and three 22-kDa thaumatin-like (TL) proteins (Pr22-1, Pr22-2, and Pr22-3). Pr22-1 and Pr22-2 were similar to the protein R class of TL proteins, whereas Pr22-3 was more similar to the S class. Pr22-3 was shown to digest laminarin, indicating that this TL protein has glucanase activity. In addition, Pr22-3 was more active in the spore bioassay than Pr22-2. Various combinations of the five proteins had a greater effect on R. secalis spores than did the individual proteins. The extraction of proteins with antifungal activity from the IWF of barley leaves indicates their possible role in defense against leaf pathogens. A similar bioassay may be developed for other systems to identify particular isoforms of pathogenicity-related proteins that might have a role in plant disease resistance.

The nucleotide-binding site of human sphingosine kinase 1.

Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly(82) of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an approximately 45-fold higher K(m)((ATP)). We have also shown that Lys(103) is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX(17-21)K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.