Palynology of the Genus Passerina (Thymelaeaceae): Relationships Form and Function

Pollen of the genus Passerina L. differs markedly from that of other southern African members of the Thymelaeaceae. Grains of most members of the Thymelaeaceae are characterised by a typical croton pattern, comprising rings of more or less trihedral sexine units mounted on an underlying reticulum of circular muri. In Passerina, however, the supratectal subunits are fused completely to form a continuous reticulum, which replaces the underlying reticulum. The reticulum in Passerina is therefore secondary in origin and not homologous with the basal reticulum of typical crotonoid grains in the family. The croton pattern has often been used as indication of a possible relationship between the Euphorbiaceae and Thymelaeaceae. Pollen of Passerina is adapted to anemophily. Grain sculpturing clearly demonstrates secondary derivation of a reticulate pattern from the typical croton pattern, through reduction, aggregation and fusion. Pollen of Passerina represents a climax of a continuum of variation in the exine of pollen in the Thymelaeaceae. As Passerina is considered phylogenetically advanced in the subfamily Thymelaeoideae, the subtribe Passerininae is raised to tribal rank, namely tribe Passerineae.     

Diurnal fluctuations in seawater pH influence the response of a calcifying macroalga to ocean acidification

Coastal ecosystems that are characterized by kelp forests encounter daily pH fluctuations, driven by photosynthesis and respiration, which are larger than pH changes owing to ocean acidification (OA) projected for surface ocean waters by 2100. We investigated whether mimicry of biologically mediated diurnal shifts in pH—based for the first time on pH time-series measurements within a kelp forest—would offset or amplify the negative effects of OA on calcifiers. In a 40-day laboratory experiment, the calcifying coralline macroalga, Arthrocardia corymbosa, was exposed to two mean pH treatments (8.05 or 7.65). For each mean, two experimental pH manipulations were applied. In one treatment, pH was held constant. In the second treatment, pH was manipulated around the mean (as a step-function), 0.4 pH units higher during daylight and 0.4 units lower during darkness to approximate diurnal fluctuations in a kelp forest. In all cases, growth rates were lower at a reduced mean pH, and fluctuations in pH acted additively to further reduce growth. Photosynthesis, recruitment and elemental composition did not change with pH, but δ¹³C increased at lower mean pH. Including environmental heterogeneity in experimental design will assist with a more accurate assessment of the responses of calcifiers to OA.     

Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.     

Structure–activity studies on the upstream splice junction of a semisynthetic intein

Protein trans-splicing by split inteins holds great potential for the chemical modification and semisynthesis of proteins. However, the structural requirements of the extein sequences immediately flanking the intein are only poorly understood. This knowledge is of particular importance for protein labeling, when synthetic moieties are to be attached to the protein of interest as seamlessly as possible. Using the semisynthetic Ssp DnaB intein both in form of its wild-type sequence and its evolved M86 mutant, we systematically varied the sequence upstream of the short synthetic Int^N fragment using both proteinogenic amino acids and unnatural building blocks. We could show for the wild-type variant that the native N-extein sequence could be reduced to the glycine residue at the (?1) position directly flanking the intein without significant loss of activity. The glycine at this position is strongly preferred over building blocks containing a phenyl group or extended alkyl chain adjacent to the scissile amide bond of the N-terminal splice junction. Despite their negative effects on the splicing yields, these unnatural substrates were well processed in the N–S acyl shift to form the respective thioesters and did not result in an increased decoupling of the asparagine cyclization step at the C-terminal splicing junction. Therefore, the transesterification step appeared to be the bottleneck of the protein splicing pathway. The fluorophore 7-hydroxycoumarinyl-4-acetic acid as a minimal N-extein was efficiently ligated to the model protein, in particular with the M86 mutant, probably because of its higher resemblance to glycine with an aliphatic c-α carbon atom at the (?1) position. This finding indicates a way for the virtually traceless labeling of proteins without inserting extra flanking residues. Due to its overall higher activity, the M86 mutant appears most promising for many protein labeling and chemical modification schemes using the split intein approach.     

Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein Complex with the t-SNARE Syntaxin 6

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Cleavage of hyaluronan is impaired in aged dermal wounds

Changes in extracellular matrix (ECM) are one of many components that contribute to impaired wound healing in aging. This study examined the effect of age on the glycosaminoglycan hyaluronan (HA) in normal and wounded dermis from young (4–6 month-old) and aged (22–24 month-old) mice. HA content and size were similar in the normal dermis of young and aged mice. Dermal explants labeled with [³H]-glucosamine showed decreased generation of smaller forms of HA in aged explants relative to young explants. Aged mice exhibited delayed wound repair compared with young mice with the greatest differential at 5 days. Expression of hyaluronan synthase (HAS) 2 and 3, and hyaluronidase (HYAL) 1–3 mRNA in wounds of young and aged mice was similar. There was a trend toward a decreased HYAL protein expression in aged wound dermis, which was accompanied by changes in detectable HYAL activity. Total HA content was similar in young and aged wound dermis. There was significantly less HA in the lower MW range (~ 250 kDa and smaller) in 5-day wound dermis, but not in 9-day wound dermis, from aged mice relative to young mice. We propose that decreased cleavage of HA is an additional component of impaired dermal wound healing in aging.     

Introduction of Asian strains and low genetic variation in farmed seaweeds: indications for new management practices

Seaweed farming has a crucial role in the development of future sustainable mariculture. In the same time, spreading of introduced species or genotypes from farms may threaten local ecosystems. We analyzed a molecular marker (mitochondrial cox2-3 spacers) from cultivated and wild specimen of the widely farmed seaweeds Eucheuma and Kappaphycus, collected in Zanzibar on the African east coast where commercial farming was introduced in 1989. Genotypes of presumed Asian origin were found growing on coral reefs and drifting in seagrass meadows, indicating that genotypes introduced for farming have established successfully in the wild in Zanzibar. Only a very low number of genotypes, all of Asian origin, were found in the farms. This indicates a low accessible gene pool, which can limit the capacity for adaptation to changed conditions and disease resistance in the farming system. African genotypes were found in a few sites, showing the potential for future farming of native strains. The ecological effects of the Asian genotypes introduced to coral reefs should also be further investigated in order to evaluate the risk connected with further introductions of new foreign strains.