Altitudinal differences in the contents of phenolics in flowering heads of three members of the tribe Lactuceae (Asteraceae) occurring as introduced species in New Zealand

Crepis capillaris, Hieracium pilosella, and Hypochaeris radicata were investigated for the influence of the altitude of the collection site on the content of phenolics within the flowering heads. These three taxa from the Lactuceae tribe of the Asteraceae family originate from Europe and are now widespread within New Zealand. Flowering heads collected from different altitudes ranging from 180 m to 1060 m (C. capillaris), from 190 to 1290 m (H. pilosella), and from 20 m to 1290 m (H. radicata), respectively, were extracted and analysed by high performance liquid chromatography. Results showed a positive correlation between the altitude of the growing site and the contents of flavonoids and phenolic acids for all investigated taxa. The altitudinal effect was, however, partially concealed by geographic differences between coastal and inland collection sites, with the inland collections containing higher concentrations of flavonoids and phenolic acids than plants collected from the coast. The results are discussed in the light of a putative UV-B protective function of the quantified compounds and of the immigration histories of the three species at hand.     

Syntheses and crystal structures of trimethyltin(IV) coordination polymers based on mixed ligands of 5-nitroisophthalate and 2,2′(4,4′)-bipy or phen

The trimethyltin(IV) polymer [(Me₃Sn)₂(nip) · EtOH]_n (1) of 5-nitroisophthalic acid (H₂nip) and its three derivatives with mixed organic N-donor ligands 2,2′-bipy [(Me₃Sn)₂(nip) · 2H₂O] · [(Me₃Sn)₂(nip) · H₂O] · 2(2,2′-bipy) (2) 4,4′-bipy {[(Me₃Sn)₂(nip)]₂(4,4′-bipy)}_n (3) or phen [(Me₃Sn)₂(nip) · H₂O] · (phen) (4) have been synthesized by the reaction of trimethyltin(IV) chloride and H₂nip when sodium ethoxide was added in the presence of 2,2′-bipy 4,4′-bipy or phen. All complexes 1–4 were characterized by elemental, IR, ¹H, ¹³C, and ¹¹⁹Sn NMR spectroscopy and X-ray crystallography analyses. Crystal, data collection and structure refinement parameters for complexes 1, 2, 3 and 4 are shown in Table 1, Table 2, respectively. The X-ray data showed the geometries of all the tin atoms in complexes 1–4 are trigonal bipyramidal. The X-ray analysis of 1 showed that the structure was a polymeric infinite chain with neighboring triorganotin centers being linked by dicarboxylate ligands and hydrogen bonds were found between adjacent chains. For 2, two different monomers were found, in one monomer, Me₃Sn were coordinated to both carboxyl groups of the ligand and water molecules were coordinated to the two Sn(IV) centers. In the other monomer, water molecules were coordinated to only one Sn center. Co-crystallized2,2′-bipy was found in 2 and a 2D supermolecular structure was formed via O–H⋯O and O–H⋯N (N atoms derived from 2,2′-bipy) hydrogen bonds. In 3 however, a 2D polymeric block was formed due to the inversion center of the 4,4′-bipy. For 4, due to the O–H⋯O and O–H⋯N (N atoms derived from phen) hydrogen bonds and intermolecular Sn⋯O bonds, a 2D polymeric structure was formed.     

The discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines (HHPQs) as potent and selective inhibitors of the mitotic kinesin-5

Here we describe the discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines (HHPQs) as potent and selective inhibitors of the mitotic kinesin-5 originally found during a high-throughput screening (HTS) campaign sampling our in-house compound collection. The compounds optimized subsequently and characterized herein were potently inhibiting the ATPase activity of Kinesin-5 and also exhibited consistent cellular activity, in that cells arrested in mitosis and apoptosis induction could be observed. X-ray crystallographic data demonstrated that these inhibitors bind in an allosteric pocket of Kinesin-5 distant from the nucleotide and microtubule binding sites. The selected clinical candidate EMD 534085 caused strong growth inhibition in human tumor xenograft models using Colo 205 colon carcinoma cells at doses below 30 mg/kg administered twice weekly without showing severe toxicity as determined by loss of body weight.     

The crystallographic structure of Panicum Mosaic Virus (PMV)

The structure of Panicum Mosaic Virus (PMV) was determined by X-ray diffraction analysis to 2.9 Å resolution. The crystals were of pseudo symmetry F23; the true crystallographic unit cell was of space group P2₁ with a = 411.7 Å, b = 403.9 Å and c = 412.5 Å, with β = 89.7°. The asymmetric unit was two entire T = 3 virus particles, or 360 protein subunits. The structure was solved by conventional molecular replacement from two distant homologues, Cocksfoot Mottle Virus (CfMV) and Tobacco Necrosis Virus (TNV), of ～20% sequence identity followed by phase extension. The model was initially refined with exact icosahedral constraints and then with icosahedral restraints. The virus has Ca⁺⁺ ions octahedrally coordinated by six aspartic acid residues on quasi threefold axes, which is completely different than for either CfMV or TNV. Amino terminal residues 1–53, 1–49 and 1–21 of the A, B and C subunits, respectively, and the four C-terminal residues (239–242) are not visible in electron density maps. The additional ordered residues of the C chain form a prominent “arm” that intertwines with symmetry equivalent “arms” at icosahedral threefold axes, as was seen in both CfMV and TNV. A 17 nucleotide hairpin segment of genomic RNA is icosahedrally ordered and bound at 60 equivalent sites at quasi twofold A–B subunit interfaces at the interior surface of the capsid. This segment of RNA may serve as a conformational switch for coat protein subunits, as has been proposed for similar RNA segments in other viruses.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Structure-based drug design of tricyclic 8H-indeno[1,2-d][1,3]thiazoles as potent FBPase inhibitors

With the goal of improving metabolic stability and further enhancing FBPase inhibitory activity, a series of tricyclic 8H-indeno[1,2-d][1,3]thiazoles was designed and synthesized with the aid of structure-based drug design. Extensive SAR studies led to the discovery of 19a with an IC₅₀ value of 1 nM against human FBPase. X-ray crystallographic studies revealed that high affinity of 19a was due to the hydrophobic interaction arising from better shape complementarity and to the hydrogen bonding network involving the side chain on the tricyclic scaffold.     

Novel 6 MV X-ray photoneutron detection and dosimetry of medical accelerators

PurposeDosimetry of fast, epithermal and thermal photoneutrons in 6 MV X-ray beams of two medical accelerators were studied by novel dosimetry methods.MethodsA Siemens ONCOR and an Elekta COMPACT medical accelerators were used. Fast, epithermal and thermal photoneutron dose equivalents in 10 cm × 10 cm 6 MV X-rays fields were determined in air and on surface of a polyethylene phantom in X and Y directions. Polycarbonate dosimeters as bare or with enriched ¹⁰B convertors (with or without cadmium covers) were used applying a 50 Hz-HV electrochemical etching method.ResultsFast, epithermal and thermal photoneutron dose equivalents were efficiently determined respectively as ∼1145.8, ∼45.3 and ∼170.6 μSv in air and ∼1888.5, ∼96.1 and ∼640.6 μSv on phantom per 100 Gy X-rays at the isocenter of Siemens ONCOR accelerator in air. The dose equivalent is maximum at the isocenter which decreases as distance from it increases reaching a constant level. Tissue-to-air ratios are constants up to 15 cm from the isocenter. No photoneutrons was detected in the Elekta COMPACT accelerator.ConclusionsFast, epithermal and thermal photoneutron dosimetry of 6 MV X-rays were made by novel dosimetry methods in a Siemens ONCOR accelerator with sum dose equivalent per Gy of ∼0.0014% μSv with ∼0.21 MeV mean energy at the isocenter; i.e. ∼150 times smaller than that of 18 MV X-rays. This observation assures clinical safety of 6 MV X-rays in particular in single-mode machines like Elekta COMPACT producing no photoneutrons due to no “beryllium exit window” in the head structure.     

Comparative structural studies of two natural isoforms of ammodytoxin,phospholipases A2 from Vipera ammodytes ammodytes which differ in neurotoxicity and anticoagulant activity

Ammodytoxin A (AtxA) and its natural isoform AtxC from the venom of Vipera ammodytes ammodytes belong to group IIA-secreted phospholipases A₂ which catalyze the hydrolysis of glycerophospholipids and exhibit strong neurotoxic and anticoagulant effects. The two isoforms, which differ in sequence by only two amino acid residues (Phe124 > Ile and Lys128 > Glu), display significant differences in toxicity and anticoagulant properties and act on protein targets including neurotoxic proteic receptors and coagulation factor Xa with significantly different strengths of binding.In order to characterize the structural basis of these functional differences, we have determined the crystal structures of the two isoforms. Comparison of the structures shows that the mutation Lys128 > Glu in AtxC could perturb interactions with FXa, resulting in lower anticoagulant activity, since the side chain of Glu128 is partly buried, making a stabilizing hydrogen bond with the main-chain nitrogen atom of residue Thr35. This interaction leads to a displacement of the main polypeptide chain at positions 127 and 128 (identified by mutagenesis as important for interaction with FXa), and a different orientation of the side chain of unmutated Lys127. The mutation Phe124 > Ile in AtxC induces no significant conformational changes, suggesting that the differences in toxicity of the two isoforms are due essentially to differences in surface complementarity in the interaction of the toxin with the neurotoxic protein receptor. The crystal structures also reveal a novel dimeric quaternary association involving significant hydrophobic interactions between the N-terminal α-helices of two molecules of ammodytoxin related by crystallographic symmetry. Interactions at the dimer interface include important contributions from Met7, which is unique to ammodytoxin. Equilibrium sedimentation experiments are consistent with the crystallographic model.Competition experiments using SPR technology show complete inhibition of AtxA binding to FXa by calmodulin (CaM). The crystal structure shows that the C-terminal region, important for binding to FXa and CaM, is fully exposed and accessible for interaction with proteic receptors in both the monomeric and dimeric forms of ammodytoxin described here.     

Electromyographic latency of postural evoked responses from the leg muscles during EquiTest Computerised Dynamic Posturography: Reference data on healthy subjects

No normative data are available for the latencies of the EMG signals from the ankle muscles in response to sudden sagittal tilt (toes-UP or toes-DOWN) or shift (shift-FOR or shift-BACK) of the support surface during standing. In this study the postural evoked response (PER) paradigm on the EquiTest™ force platform was applied to 31 healthy adults (18 women and 13 men; mean age 29 years). The EMG latencies (PEREMG) were computed both through the standard manual procedure and through a specially designed automated algorithm. The manually computed PEREMG onset yielded a 95% tolerance interval between 82 ms and 148 ms after toes-UP perturbation, between 93 ms and 182 ms after toes-DOWN perturbation, between 67 ms and 107 ms after shift-BACK perturbation, and between 73 ms and 113 ms after shift-FOR perturbation. When comparing the two methods, paired t-tests showed no significant mean difference (Bonferroni-adjusted p-values ranged from 0.440 to 1.000) and all Bland–Altman plots included zero difference within the limits of agreement. Therefore, the manual and the automated methods appear to be sufficiently consistent. These results foster the clinical application of PEREMG testing on the EquiTest platform.     

Variation in the ribosomal ITS-sequence of the lichens Buellia frigida and Xanthoria elegans from the Vestfold Hills,eastern Antarctica

Abstract:Thalli of the lichens Buellia frigida and Xanthoria elegans were collected from five different locations each 5–15 km apart in the Vestfold Hills, Princess Elizabeth Land, eastern Antarctica. A further collection was made from Mawson Station, Mac Robertson Land, eastern Antarctica, 660 km away. DNA was extracted from whole thalli and the ribosomal ITS region amplified by PCR using fungal specific primers. Resulting products were sequenced to gain an indication of whether or not variation was present within populations of lichen-forming fungi from continental Antarctica, and therefore of the availability of genetic resources to react to pressures such as climate change. Three genotypes ofB. frigida and two of X. elegans were detected in the Vestfold Hill collections. However, these differed by only one nucleotide position suggesting the presence of relatively little genetic variation, if the ITS region is indicative of the overall genome. Buellia frigida collected from Mawson Station had an identical ITS region sequence to the most common Vestfold Hills genotype, indicating that this species may have a low level of genetic variation across much of eastern Antarctica. In contrast, X. elegans collected from Mawson showed considerable genetic variation from the Vestfolds thalli, differing at 14·2% of nucleotide positions and had an identical ITS region sequence to an isolate from maritime Antarctica 4960 km away. Samples from the Vestfold Hills formed a distinct cluster in a phylogenetic analysis of ITS sequences from a worldwide collection of X. elegans isolates.     

A critical evaluation of salivary testosterone as a method for the assessment of serum testosterone

Although salivary testosterone (T) is often used in clinical studies accuracy is mostly questionable. State of the art data for men is sparse and for women absent. Our objective was to perform a critical evaluation of salivary T (Sal-T) as a method for indirect assessment of serum T using state of the art methods. Saliva was collected via ‘Salivette’ and ‘passive drooling’ methods. Sal-T and free T in serum after equilibrium dialysis were measured by LC-MS/MSResultsEvaluation of Sal-T results versus free T by equilibrium dialysis (ED-T) for men gave: ‘Salivette’ Sal-T = 0.05 + 0.88x ED-T, r = 0.43; ‘passive drooling’ Sal-T = 0.17 + 0.91x ED-T r = 0.71. In women, correlation was comparable but values are higher than free T: ‘passive drooling’ Sal-T = 0.12 + 2.32x ED-T, r = 0.70. The higher than expected T values in saliva, appear to be explained by T binding to salivary proteins. Iso-electric focusing of saliva proteins, followed by fractionation and LC–MS/MS assay of T showed marked testosterone peaks at pH 5.3 and 8.4, providing evidence for T binding in saliva to proteins such as albumin and proline rich protein (PRP).ConclusionsPassive drooling is the collection method of choice for testosterone in saliva. Sal-T is not directly comparable to serum free T due to T binding to saliva proteins, which substantially affects the low Sal-T in women but not the higher Sal-T in healthy adult men.     

The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy

Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving > 3.5 Å resolution detail in membrane proteins of modest (~ 300 kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function.     

Structure-based design of novel HCV NS5B thumb pocket 2 allosteric inhibitors with submicromolar gt1 replicon potency: Discovery of a quinazolinone chemotype

We describe the structure-based design of a novel lead chemotype that binds to thumb pocket 2 of HCV NS5B polymerase and inhibits cell-based gt1 subgenomic reporter replicons at sub-micromolar concentrations (EC₅₀ <200 nM). This new class of potent thumb pocket 2 inhibitors features a 1H-quinazolin-4-one scaffold derived from hybridization of a previously reported, low affinity thiazolone chemotype with our recently described anthranilic acid series. Guided by X-ray structural information, a key NS5B–ligand interaction involving the carboxylate group of anthranilic acid based inhibitors was replaced by a neutral two-point hydrogen bonding interaction between the quinazolinone scaffold and the protein backbone. The in vitro ADME and in vivo rat PK profile of representative analogs are also presented and provide areas for future optimization of this new class of HCV polymerase inhibitors.     

Decreased fibularis reflex response during inversion perturbations in FAI subjects

Investigate reflex responses in muscles throughout the lower limb and low back during sudden inversion perturbations in individuals with and without Functional Ankle Instability (FAI) while walking. Forty subjects participated in the study. Surface electromyogram recordings were obtained from the fibularis (FIB), gluteus medius (GM), erector spinae (ES), and sternocleidomastoid (SCM) of the injured/matched side as well as the uninjured/matched contralateral side (FIB_CLS, GM_CLS, or ES_CLS). Latency and amplitude data were collected while subjects were walking on a custom-built perturbation walkway. The onset of the short-latency stretch reflex of the FIB was significantly later in the injured side of the FAI individuals when compared to the control group (P = 0.009). Both the short and long latency reflex amplitude was significantly smaller in the FIB muscle in the FAI group than in the control group (P < 0.008). No significant differences in latency or amplitude reflex responses were identified between the two groups in the GM, ES, FIB_CLS, GM_CLS, or ES_CLS (P > .05). Interpretation of these results indicate that during a dynamic perturbation task individuals with FAI demonstrate longer fibularis muscle latencies on the injured side while no significant changes in the proximal muscle groups. Additionally, short and long latency reflex amplitude was significantly decreased in FAI individuals.     

Sampling of Bemisia tabaci adults using a pre-programmed autonomous pest control robot

An autonomous robot (Cabbot) was built to control insect pests in a plastic greenhouse and utilized to sample adult whiteflies (Bemisia tabaci) on paprika plants (Capsicum annum var. angulosum). To accomplish this, a sampling device consisting of an air compressor for pest agitation, a sticky trap (100 mm × 150 mm) and an image processing system for pest identification were installed on the Cabbot. The sampling precision of the Cabbot (D = 0.16) was higher than that of the sticky trap (D = 0.19) when sampling of adult white flies was conducted in caged pots. The Cabbot could also collect a substantial number of individuals within a short duration (3 minutes). The collection efficiency (i.e., percent of samples collected at least n (1 in this case) individuals to the total sample number) of insects by the Cabbot was markedly high at low population size, showing approximately 30% when the population size was ≤ 16 individuals per plant. The sampling precision and collection efficiency suggested that the Cabbot is effective in local, short-term sampling and could be used for early warning of pest occurrences.     

A novel position-sensitive mega-size dosimeter for photoneutrons in high-energy X-ray medical accelerators

PurposeA novel position-sensitive mega-size polycarbonate (MSPC) dosimeter is introduced. It provides photoneutron (PN) dose equivalent matrix of positions in and out of a beam of a high energy X-ray medical accelerator under a single exposure.MethodsA novel position-sensitive MSPC dosimeter was developed and applied. It has an effective etched area of 50 × 50 cm², as used in this study, processed in a mega-size electrochemical etching chamber to amplify PN-induced-recoil tracks to a point viewed by the unaided eyes. Using such dosimeters, PN dose equivalents, dose equivalent profiles and isodose equivalent distribution of positions in and out of beams for different X-ray doses and field sizes were determined in a Siemens ONCOR Linac.ResultsThe PN dose equivalent at each position versus X-ray dose was linear up to 20 Gy studied. As the field size increased, the PN dose equivalent in the beam was also increased but it remained constant at positions out of the beam up to 20 cm away from the beam edge. The jaws and MLCs due to material differences and locations relative to the target produce different PN contributions.ConclusionsThe MSPC dosimeter introduced in this study is a perfect candidate for PN dosimetry with unique characteristics such as simplicity, efficiency, dose equivalent response, large size, flexibility to be bent, resembling the patient’s skin, highly position-sensitive with high spatial resolution, highly insensitive to X-rays, continuity in measurements and need to a single dosimeter to obtain PN dose equivalent matrix data under a single X-ray exposure.     

The design and optimization of a series of 2-(pyridin-2-yl)-1H-benzimidazole compounds as allosteric glucokinase activators

The optimization of a series of benzimidazole glucokinase activators is described. We identified a novel and potent achiral benzimidazole derivative as an allosteric GK activator. This activator was designed and synthesized via removal of the chiral center of the lead compound, 6-(N-acylpyrrolidin-2-yl)benzimidazole. The activator exhibited good PK profiles in rats and dogs, and significant hypoglycemic efficacy at 1 mg/kg po dosing in a rat OGTT model. The binding site and binding mode of the benzimidazole class of GKA with GK protein was confirmed by X-ray crystallographic analysis.     

Synthesis,SAR, and X-ray structure of tricyclic compounds as potent FBPase inhibitors

With the aim of discovering a novel class of fructose-1,6-bisphosphatase (FBPase) inhibitors, a series of compounds based on tricyclic scaffolds was synthesized. Extensive SAR studies led to the finding of 8l with an IC₅₀ value of 0.013 μM against human FBPase. An X-ray crystallographic study revealed that 8l bound at AMP binding sites of human liver FBPase with hydrogen bonding interactions similar to AMP.     

Fruit nutritional composition and non-nutritive traits of indigenous South African tree species

Frugivorous animals play a major role in dispersing tropical, and to a lesser extent, temperate tree species. In order to attract potential seed dispersers, plants generally offer a reward of fleshy fruit pulp. Criteria for fruit choice by avian frugivores are influenced by a number of non-nutritive (e.g. fruit size and colour) factors; and nutritional composition of the fruit. There is a paucity of nutritional composition and other fruit trait data of indigenous South African fruit. This information is necessary in order to determine which frugivores are likely to ingest which fruits and consequently act as potential seed dispersal agents. This information would provide us with an understanding of the inter-relationships between indigenous fruit and frugivores in South Africa. Consequently nutritional composition was investigated in various indigenous fruit species that avian frugivores feed on. Fruits were collected from 38 indigenous tree species found in KwaZulu-Natal Afromontane and coastal forests. Pulp was freeze-dried to constant mass and then analysed for sugar, lipid and protein content; and for water content determination. Fruit width in this study ranged from 4 mm (Searsia rehmanniana and Trema orientalis) to 40 mm (Annona senegalensis, Ficus sur and Xylotheca kraussiana). Of the fruits examined in this study 29% were black and 43% were red when ripe. Most (84%) fruit species analysed for sugar content were hexose dominant with 50% being fructose and 34% being glucose dominant. Only 16% of the fruit species analysed were sucrose dominant. Fruits in this study were generally observed to be high (mean: 68.1 ± 3.3%; n = 30) in water content; and low in protein and lipid content respectively (mean: 8.2 ± 0.5%; 9.3 ± 2.2%; n = 30) indicating that these fruit species could be considered as nutrient-dilute. Future studies need to determine the nutritional composition of the remaining indigenous South African fruit in order to develop a comprehensive database as well as examining non-nutritive factors.     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.