Anhydrobiosis and Freezing-Tolerance: Adaptations That Facilitate the Establishment of Panagrolaimus Nematodes in Polar Habitats

Anhydrobiotic animals can survive the loss of both free and bound water from their cells. While in this state they are also resistant to freezing. This physiology adapts anhydrobiotes to harsh environments and it aids their dispersal. Panagrolaimus davidi, a bacterial feeding anhydrobiotic nematode isolated from Ross Island Antarctica, can survive intracellular ice formation when fully hydrated. A capacity to survive freezing while fully hydrated has also been observed in some other Antarctic nematodes. We experimentally determined the anhydrobiotic and freezing-tolerance phenotypes of 24 Panagrolaimus strains from tropical, temperate, continental and polar habitats and we analysed their phylogenetic relationships. We found that several other Panagrolaimus isolates can also survive freezing when fully hydrated and that tissue extracts from these freezing-tolerant nematodes can inhibit the growth of ice crystals. We show that P. davidi belongs to a clade of anhydrobiotic and freezing-tolerant panagrolaimids containing strains from temperate and continental regions and that P. superbus, an early colonizer at Surtsey island, Iceland after its volcanic formation, is closely related to a species from Pennsylvania, USA. Ancestral state reconstructions show that anhydrobiosis evolved deep in the phylogeny of Panagrolaimus. The early-diverging Panagrolaimus lineages are strongly anhydrobiotic but weakly freezing-tolerant, suggesting that freezing tolerance is most likely a derived trait. The common ancestors of the davidi and the superbus clades were anhydrobiotic and also possessed robust freezing tolerance, along with a capacity to inhibit the growth and recrystallization of ice crystals. Unlike other endemic Antarctic nematodes, the life history traits of P. davidi do not show evidence of an evolved response to polar conditions. Thus we suggest that the colonization of Antarctica by P. davidi and of Surtsey by P. superbus may be examples of recent “ecological fitting” of freezing-tolerant anhydrobiotic propagules to the respective abiotic conditions in Ross Island and Surtsey.     

A non‐helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition

MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na⁺‐dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate‐bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly‐Met‐Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.     

Stereoselective binding of benzodiazepines and related compounds on calmodulin-sepharose

A method has been presented to evaluate the Ca²⁺-dependent binding affinity of ligands to calmodulin (CaM) from their chromatographic retentions obtained on a CaM-Sepharose column. Enantiomers of benzodiazepines and related compounds could be separated. Among chiral calcium antagonists, verapamil showed stereoselectivity of binding. © 1995 Wiley-Liss, Inc.     

A new linear cyclin docking motif that mediates exclusively S‐phase CDK‐specific signaling

Cyclin‐dependent kinases (CDKs), the master regulators of cell division, are activated by different cyclins at different cell cycle stages. In addition to being activators of CDKs, cyclins recognize various linear motifs to target CDK activity to specific proteins. We uncovered a cyclin docking motif, NLxxxL, that contributes to phosphorylation‐dependent degradation of the CDK inhibitor Far1 at the G1/S stage in the yeast Saccharomyces cerevisiae. This motif is recognized exclusively by S‐phase CDK (S‐CDK) Clb5/6‐Cdc28 and is considerably more potent than the conventional RxL docking motif. The NLxxxL and RxL motifs were found to overlap in some target proteins, suggesting that cyclin docking motifs can evolve to switch from one to another for fine‐tuning of cell cycle events. Using time‐lapse fluorescence microscopy, we show how different docking connections temporally control phosphorylation‐driven target degradation. This also revealed a differential function of the phosphoadaptor protein Cks1, as Cks1 docking potentiated degron phosphorylation of RxL‐containing but not of NLxxxL‐containing substrates. The NLxxxL motif was found to govern S‐cyclin‐specificity in multiple yeast CDK targets including Fin1, Lif1, and Slx4, suggesting its wider importance.     

Effects of neonicotinoid seed treatments on wild bee populations in soybean and corn fields in eastern Ontario

1. Neonicotinoid-coated corn and soybean seeds are a common crop in Canada and the US. A growing body of research is demonstrating that, through various exposure routes, neonicotinoids can impact a suite of nontarget organisms including beneficial insects such as bees. However, to date, only a few studies have examined the effects of neonicotinoids in field settings.
2. We assessed the relationship between agricultural crop soil neonicotinoid levels and wild bee abundance and diversity at 16 agricultural sites representing different soil neonicotinoid levels. We detected clothianidin at 11 sites, thiamethoxam at three sites; imidacloprid was not detected.
3. Hedgerow and crop soils were consistent in terms of where clothianidin was detected; thiamethoxan was not detected in hedgerow soils. Based on model outcomes, fields with higher levels of soil neonicotinoids exhibited significantly lower wild bee abundance and diversity than those with low or no neonicotinoids detected.
4. Crop soil neonicotinoid level, hedgerow floral resource abundance and crop type were consistent predictors of bee abundance across models; only neonicotinoid level and crop type were significant predictors of diversity.
5. Our results are consistent with recent findings in the midwestern US, and underscore the potential risk of soil neonicotinoids to wild bee populations across regions and crop systems.

     

Peptide based vaccine design: synthesis and immunological characterization of branched polypeptide conjugates comprising the 276-284 immunodominant epitope of HSV-1 glycoprotein D.

The importance of the length and conjugation site of a protective epitope peptide (276SALLEDPVG284) from glycoprotein D of herpes simplex virus in branched polypeptide conjugates has been investigated. A new set of peptides, with a single attachment site and truncated sequences, was prepared. The immunogenicity of conjugates and the specificity of antibody responses elicited were investigated in BALB/c, C57/B1/6 and CBA mice. It was found that the covalent coupling of the peptide comprising the 276-284 sequence of gD through its Asp residue at position 281 did not influence the immunogenic properties of the epitope, while involvement of the side chain of Glu at position 280 almost completely abolished immunogenicity. These results clearly indicated that the conjugation site of the epitope peptide influenced the intensity and specificity of antibody responses. Comparison of the immunological properties of conjugates containing truncated gD peptides revealed the presence of two epitopes within the 276-284 region. One of the proposed epitopes is situated at the N-terminal (276-281) region, while the other is located at the C-terminal end of the sequence (279-284). Binding data demonstrated that some of the peptides comprising these epitopes induced gD-specific responses in their conjugated form and also elicited an immune response that conferred protection against lethal HSV-1 infection. The correlation of peptide- and gD-specific antibody responses with the protective effect of the immune response is discussed.