Isolation and partial characterization of 3 nontoxic d‐galactose–specific isolectins from seeds of Momordica balsamina

Three isolectins denoted hereforth MBaL‐30, MBaL‐60, and MBaL‐80 were isolated from seeds extract of Momordica balsamina by 30%, 60%, and 80% ammonium sulfate saturations, respectively. The native molecular weights of these lectins, as judged by gel filtration, were 108, 56, and 160 kDa, respectively. On SDS‐PAGE, under reduced condition, 27 kDa band was obtained for all isolectins. The lectins hemagglutinating activities were variably inhibited by d ‐galactose (minimum inhibitory concentrations = 12.5mM, 50mM, and 0.391mM, respectively). MBaL‐30 and ‐60 could agglutinate all human blood types with slight preference for the A and O blood groups, whereas MBaL‐80 did not agglutinate B and AB blood types. The 3 isolectins were purified from crude seeds extract, collectively, in a single step on the affinity matrix Lactamyl‐Seralose 4B; this purified lectin fraction, which contains all isolectins, is termed MBaL. The N‐terminal of MBaL till the 25th amino acid was NLSLSELDFSADTYKSFIKNLRKQL, which shares 88% sequence identity with Momordica charantia lectin type‐2 ribosomal inactivating protein from Momordica charantia and 50% with momordin II from Momordica balsamina . MBaL retained 100% activity at up to 50°C for 30 minutes. MBaL‐30 and MBaL‐60 exhibited maximum activities in the pH range between 4 and 8, while MBaL‐80 was showing maximum activity in the pH range between 3 and 5. Treatment of MBaL‐30 and MBaL‐60 with EDTA completely abolished their hemagglutinating activities. Addition of Zn and Fe ions to the ethylenediaminetetraacetic acid–treated MBaL‐30 and MBaL‐60 lectins did not only regained the loss of activity but also resulted in 200% to 300% increase in activity, respectively. MBaL‐30 and ‐60 agglutinated gram positive Listeria monocytogenes and Staphylococcus aureus, whereas MBaL‐30 could merely agglutinate Escherichia coli . None of these lectins could arrest bacterial growth. Addition of MBaL to cancer cell lines (Gastric cancer cell line (AGS) and Gastric cencer cell line (MKN45), Glioblastoma (ECV‐304), and Human urinary bladder cancer cell line (U87‐MG)) at varying concentrations did not cause statistically significant changes on cell growth and viability.     

Proteomic profiling of lymphedema development in mouse model

The lymphatic vascular system plays an important role in tissue fluid homeostasis. Lymphedema is a chronic, progressive, and incurable condition that leads to lymphatic fluid retention; it may be primary (heritable) or secondary (acquired) in nature. Although there is a growing understanding of lymphedema, methods for the prevention and treatment of lymphedema are still limited. In this study, we investigated differential protein expressions in sham‐operated and lymphedema‐operated mice for 3 days, using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry analysis. Male improved methodology for culturing noninbred (ICR) mice developed lymphedema in the right hindlimb. Twenty functional proteins were found to be differentially expressed between lymphedema induced‐right leg tissue and normal left leg tissue. Out of these proteins, the protein levels of apolipoprotein A‐1 preprotein, alpha‐actinin‐3, mCG21744, parkinson disease, serum amyloid P‐component precursor, annexin A8, mKIAA0098 protein, and fibrinogen beta chain precursor were differentially upregulated in the lymphedema mice compared with the sham‐operated group. Western blotting analysis was used to validate the proteomics results. Our results showing differential up‐regulation of serum amyloid P‐component precursor, parkinson disease, and apolipoprotein A‐1 preprotein in lymphedema model over sham‐operated model suggest important insights into pathophysiological target for lymphedema. Copyright © 2016 John Wiley & Sons, Ltd.     

The functions of foliar nyctinasty: a review and hypothesis

Foliar nyctinasty is a plant behaviour characterised by a pronounced daily oscillation in leaf orientation. During the day, the blades of nyctinastic plant leaves (or leaflets) assume a more or less horizontal position that optimises their ability to capture sunlight for photosynthesis. At night, the positions that the leaf blades assume, regardless of whether they arise by rising, falling or twisting, are essentially vertical. Among the ideas put forth to explain the raison d'être of foliar nyctinasty are that it: (i) improves the temperature relations of plants; (ii) helps remove surface water from foliage; (iii) prevents the disruption of photoperiodism by moonlight; and (iv) directly discourages insect herbivory. After discussing these previous hypotheses, a novel tritrophic hypothesis is introduced that proposes that foliar nyctinasty constitutes an indirect plant defence against nocturnal herbivores. It is suggested that the reduction in physical clutter that follows from nocturnal leaf closure may increase the foraging success of many types of animals that prey upon or parasitise herbivores. Predators and parasitoids generally use some combination of visual, auditory or olfactory cues to detect prey. In terrestrial environments, it is hypothesised that the vertical orientation of the blades of nyctinastic plants at night would be especially beneficial to flying nocturnal predators (e.g. bats and owls) and parasitoids whose modus operandi is death from above. The movements of prey beneath a plant with vertically oriented foliage would be visually more obvious to gleaning or swooping predators under nocturnal or crepuscular conditions. Such predators could also detect sounds made by prey better without baffling layers of foliage overhead to damp and disperse the signal. Moreover, any volatiles released by the prey would diffuse more directly to the awaiting olfactory apparatus of the predators or parasitoids. In addition to facilitating the demise of herbivores by carnivores and parasitoids, foliar nyctinasty, much like the enhanced illumination of the full moon, may mitigate feeding by nocturnal herbivores by altering their foraging behaviour. Foliar nyctinasty could also provide a competitive advantage by encouraging herbivores, seeking more cover, to forage on or around non‐nyctinastic species. As an added advantage, foliar nyctinasty, by decreasing the temperature between plants through its effects on re‐radiation, may slow certain types of ectothermic herbivores making them more vulnerable to predation. Foliar nyctinasty also may not solely be a behavioural adaptation against folivores; by discouraging foraging by granivores, the inclusive fitness of nyctinastic plants may be increased.     

Host selection as a downstream strategy: polyelectrolyte precipitation of beta-glucuronidase from plant extracts.

Host selection can be a strategy to simplify downstream processing for protein recovery. Advancing capabilities for using plants as hosts offers new host opportunities that have received only limited attention from a downstream processing perspective. Here, we investigated the potential of using a polycationic precipitating agent (polyethylenimine; PEI) to precipitate an acidic model protein (beta-glucuronidase; GUS) from aqueous plant extracts. To assess the potential of host selection to enhance the ease of recovery, the same procedure was applied to oilseed extracts of canola, corn (germ), and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein were investigated--the wild-type enzyme (WTGUS) and a genetically engineered version containing 10 additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola was the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (30 mg PEI/g total protein), and over 80% of the initial WTGUS activity was recovered with 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS but only 1.3-fold enrichment. Corn, although requiring the most PEI relative to total protein to precipitate (210 mg PEI/g total protein for 100% precipitation), gave intermediate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yields of recoverable activity and greater purification factors.     

The evolutionary geometry of human anatomy: Discovering our inner fly

The human body is one still frame in a very long evolutionary movie. Anthropologists focus on the last few scenes, whereas geneticists try to trace the screenplay back as far as possible. Despite their divergent time scales (millions versus billions of years), both disciplines share a reliance on a third field of study whose scope spans only a matter of days to months, depending on the organism. Embryology is crucial for understanding both the pliability of anatomy and the modularity of gene circuitry. The relevance of human embryology to anthropology is obvious. What is not so obvious is the notion that equally useful clues about human anatomy can be gleaned by studying the development of the fruit fly, an animal as different from us structurally as it is distant from us evolutionarily. The underlying kinship between ourselves and flies has only become apparent recently, thanks to revelations from the nascent field of evolutionary developmental biology, or evo‐devo. All bilaterally symmetric animals, it turns out, share a common matrix of body axes, a common lexicon of intercellular signals, and a common arsenal of genetic gadgetry that evolution has tweaked in different ways in different lineages to produce a dazzling spectrum of shapes and patterns. Anthropologists can exploit this deep commonality to search our genome more profitably for the mutations that steered us so far astray from our fellow apes.     

Architecture and function of metallopeptidase catalytic domains

The cleavage of peptide bonds by metallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in all major physiological processes, and so their deregulation leads to diseases ranging from cancer and metastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders. MPs cleave their substrates without a covalent intermediate in a single‐step reaction involving a solvent molecule, a general base/acid, and a mono‐ or dinuclear catalytic metal site. Most monometallic MPs comprise a short metal‐binding motif (HEXXH), which includes two metal‐binding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe of MPs. The latter divides mainly into the gluzincin and metzincin clans. Metzincins consist of globular ～130–270‐residue catalytic domains, which are usually preceded by N‐terminal pro‐segments, typically required for folding and latency maintenance. The catalytic domains are often followed by C‐terminal domains for substrate recognition and other protein–protein interactions, anchoring to membranes, oligomerization, and compartmentalization. Metzincin catalytic domains consist of a structurally conserved N‐terminal subdomain spanning a five‐stranded β‐sheet, a backing helix, and an active‐site helix. The latter contains most of the metal‐binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C‐terminal subdomains are generally shorter, differ more among metzincins, and mainly share a conserved loop—the Met‐turn—and a C‐terminal helix. The accumulated structural data from more than 300 deposited structures of the 12 currently characterized metzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of their working mechanisms, and form the basis for the design of novel drugs.