Embryo lipoproteins and yolk lipovitellin consumption during embryogenesis in Macrobrachium borellii (Crustacea: Palaemonidae)

The prawn Macrobrachium borellii has lecithotrophic eggs with highly-abbreviated development. The major yolk component is lipovitellin (LV), a lipoprotein with 30% lipids (by weight). LV consumption during embryogenesis was followed by ELISA and Western blot analysis using an anti-LV polyclonal antibody. No cross-reacting proteins were observed and LV-like lipoproteins were strongly recognized by the antibody in hemolymph (vitellogenin), yolk (LV) and embryos (LVe), as determined by Western Blot analysis. LV decreased significantly along development from 9.4 to 1.1 microg/mg egg. Consumption rate of LV was slow in early embryogenesis, followed by a rapid utilization in late embryonic stages. Significant LVe amounts were still present at hatching. LV apolipoproteins were selectively degraded during embryo development, being the highest molecular weight subunit the most affected. Comparison among in vitro, in vivo and theoretical proteolysis suggested that trypsin may be involved in LV degradation during late embryogenesis. Embryo lipoprotein (HDLe) synthesis was first detected at stage 6. HDLe shared the same density, MW and subunit composition as adult hemolymph HDL(1) and did not cross-react with LV-like lipoproteins. Though expressed at low concentration, it fulfilled embryo needs for lipid transport among organs.     

Influence of host plant nitrogen fertilization on hemolymph protein profiles of herbivore Spodoptera exigua and development of its endoparasitoid Cotesia marginiventris

Nitrogen has complex effects on plant–herbivore–parasitoid tritrophic interactions. The negative effects of low nitrogen fertilization in host plants on insect herbivores can be amplified to the higher trophic levels. In the present study, we examined the impact of varying nitrogen fertilization (42, 112, 196, and 280 ppm) of cotton plants (Gossypium hirsutum L.) on the interactions between the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), and the hymenopteran endoparasitoid Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae). We predicted that the development and fitness of C. marginiventris would be adversely affected by low host plant nitrogen fertilization through the herbivore S. exigua. The percentage of C. marginiventris offspring developing to emerge and spin a cocoon, and total mortality of parasitized S. exigua larvae were unaffected by nitrogen level. The developmental time of C. marginiventris larvae in S. exigua larvae feeding on low (42 ppm) nitrogen cotton plants was approximately 30% longer than that of those feeding on higher (112, 196, and 280 ppm) nitrogen plants. Parasitoid size (length of right metathoracic tibia), a proxy for fitness, of C. marginiventris males was positively affected by nitrogen level. Total amounts of S. exigua hemolymph proteins were not affected by nitrogen level, but were reduced by parasitism by C. marginiventris. Two proteins with molecular weights of ca. 84 and 170 kDa dominated the S. exigua larval hemolymph proteins. Concentrations of the 170 kDa hemolymph protein were unaffected by nitrogen treatment, but parasitism reduced concentrations of the 170 kDa protein. Concentrations of the 84 kDa protein, on the other hand, were interactively affected by parasitism and nitrogen treatment: higher nitrogen fertilization (112, 196, and 280 ppm) increased protein concentrations relative to the 42 ppm treatment for unparasitized S. exigua larvae, whereas nitrogen treatment had no effects on parasitized larvae. For S. exigua larvae feeding on 42 ppm nitrogen plants, parasitism increased concentration of the 84 kDa protein, while for those feeding on 112, 196, and 280 ppm nitrogen plants, parasitism decreased concentrations of the protein. Possible mechanisms and ecological consequences for the extended development of C. marginiventris on S. exigua hosts grown on low-nitrogen plants are discussed.     

The effect of developmental stage on eggshell thickness variation in endangered falcons

We compared eggshell thickness of hatched eggs with that of non-developed eggs in endangered falcon taxa to explore the effect of embryo development on eggshell thinning. To our knowledge, this has never been examined before in falcons, despite the fact that eggshell thinning due to pollutants and environmental contamination is often considered the most common cause of egg failure in falcons. Because of the endangered nature of these birds, and the difficulty in gaining access to the nests and their eggs, there is a large gap in our knowledge regarding eggshell thickness variation and the factors affecting it. We used a linear mixed-effects (LME) model to explore the variation in eggshell thickness (n = 335 eggs) in relation to the developmental stage of the eggs, but also in relation to the falcon taxa, the laying sequence and the study zone. Female identity (n = 69) and clutch identity (n = 98) were also included in the LME model. Our results are consistent with the prediction that eggshell thickness decreases during incubation because of the important effect of calcium uptake by the embryo during development. Our results also show that eggs laid later in the sequence had significantly thinner eggshells. In this study, we provide the first quantitative data on eggshell thickness variation of hatched eggs in different falcon taxa that were not subjected to contamination or food limitation (i.e., bred under captive conditions). Because eggshell thickness strongly influences survival and because the species examined in this study are endangered, our data represent a valuable control for future studies on the effects of pollution on eggshells from wild populations and thus are an important contribution to the conservation of falcons.     

Calcium protection of PS2 complex of Phaseolus coccineus from cadmium toxicity: In vitro study

The action of 10 and 20 mM Ca against harmful Cd effect on PS2 complex isolated from leaves of Phaseolus coccineus L. cv. Pi?kny Ja? was studied. The changes in fast chlorophyll a fluorescence induction kinetics and protein composition of PS2 complex were the symptoms of Cd toxicity and Ca protection of PS2 complex. Calcium applied at 10 mM concentration prevented F₀ reduction caused by the presence of 250–1000 μM Cd in the incubation mixture, but that of (the variable chlorophyll a fluorescence) F_v, F_m, F_v/F₀, and F_v/F_m only at 250 μM Cd. Ca concentration doubling in the incubation mixture resulted in complete overcoming the toxicity of 250–1000 μM Cd to F_v and F_m. However, the protection of F_v/F₀ and the photochemical efficiency of PS2 (F_v/F_m) from 1000 μM Cd was only partial even at 20 mM Ca. A protective effect of 10 mM Ca on D1, D2 and 17 kDa proteins was found in PS2 complex exposed to 250 μM Cd, and on 43 kDa protein in the complex treated with 500 μM Cd. However, 20 mM Ca counteracted the toxicity of 500 μM Cd to the 43, 47 and 17 kDa proteins, as well as the harmful effect of 1000 μM Cd on 47 and 17 kDa ones.     

Differential regulation of extracellular matrix constituents in myocardial remodeling with and without heart failure following pressure overload

Patients with aortic stenosis develop various degrees of myocardial hypertrophy and heart failure (HF) despite comparable transvalvular gradients. An important element in the transition from compensated hypertrophy to HF is dilatation of the left ventricle (LV). The molecular pathology associated with LV dilatation and development of HF is not known. Thus, we examined potential differences in the regulation of myocardial extracellular matrix (ECM) constituents in mice with hypertrophy only (ABnonHF) and with HF (ABHF) as response to comparable pressure overload. The ascending aorta was banded, or left loose in sham-operated mice. Increased lung weight and left atrial diameter indicating pulmonary congestion were used to identify ABHF mice. Cardiac function and geometry were evaluated by echocardiography. Despite comparable pressure gradients and cardiac output, ABHF had reduced fractional shortening (23%), reduced systolic (28%) and diastolic (32%) tissue velocity and increased LV internal dimension in diastole (10%) and systole (17%) (LVIDd/s) compared to ABnonHF (p ≤ 0.05). Microarray analyses identified 120 differently regulated genes related to ECM in ABHF compared to ABnonHF (p ≤ 0.05). Interestingly, in ABHF, we found a 24% (p ≤ 0.05) reduction of the LV collagen VIII protein levels despite increased levels of LV total collagen by 23% (p ≤ 0.05). LV collagen VIII correlated negatively with LVIDd (R = 0.55, p = 0.03) and LVIDs (R = 0.72, p = 0.002). As this protein may function as a “sealant” binding collagen fibrils together, reduction of collagen VIII could potentially contribute to LV dilatation and development of HF.     

Immunodetection and immunolocalization of tryptophanins in oat (Avena sativa L.) seeds

Tryptophanins (TRPs) are low molecular weight, tryptophan-rich, basic proteins found in oat (Avena sativa L.) seeds. Like their counterpart puroindolines (PINs) from wheat (Triticum aestivum L.), TRPs are thought to be involved in flour softness as well as disease resistance against phytopathogenic fungi. PINs are known to be the major components of ‘friabilin’ associated with the surface of water washed starch grains and possess lipid binding properties. Two polyclonal antisera against puroindoline-a (PIN-a), and puroindoline-b (PIN-b) respectively; and a monoclonal antiserum raised against ‘friabilin’ were used as primary antibodies in immunoblotting experiments. All antisera detected immunoreactive polypeptides, with approximate relative masses of 15–16 kDa, in oat, wheat, and barley (Hordeum vulgare L.) seed extracts but not in rice (Oryza sativa L.), maize (Zea mays L.), bean (Phaseolus vulgaris L.), pea (Pisum sativum L.) and lentil (Lens culinaris Medic.) seed extracts. Immunoreactive polypeptides were detected in aqueous ethanol [52% (v/v) ethanol] seed extracts. Both anti-‘friabilin’ monoclonal and anti-PIN-b polyclonal antisera recognized 15 as well as 16 kDa tryptophanins in oat seeds from different cultivars. On the other hand, anti-PIN-a polyclonal antiserum strongly cross-reacted with 16 kDa TRP and weakly with 15 kDa TRP. Tryptophanins were found to be associated with the surface of starch grains in oat endosperm tissue using both fluorescence and confocal laser scanning microscopies with anti-‘friabilin’ monoclonal antiserum. SDS-PAGE and immunoblotting assays revealed a gradual synthesis of TRPs as early as milk stage in developing oat seeds. On the other hand, TRPs tend to undergo degradation during seed germination.     

Regulation of plasma membrane Ca2 +-ATPase activity by acetylated tubulin: Influence of the lipid environment

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca^2 +-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300 μg/ml) in combination with acidic lipids at concentrations > 10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50 μg/ml tubulin, increased PMCA activity > 12-fold, whereas tubulin alone at high concentration (≥ 300 μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca^2 + transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (< 50 μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.     

Oviposition and reproductive performance of a generalist parasitoid (Trichogramma pretiosum) exposed to host species that differ in their physical characteristics

The response of generalist egg parasitoids to alternative natural hosts that are present simultaneously is not well known. We investigated the behavior of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) in relation to two field hosts Helicoverpa armigera Hübner and Spodoptera litura Fabricius, in choice and no choice tests. We quantified the effects of natal host species and post-emergence adult age on the oviposition preference of the parasitoids. H. armigera eggs were consistently preferred over S. litura eggs, regardless of the natal host and adult age. When only S. litura eggs were available as hosts, they were parasitized at statistically similar rates to H. armigera eggs (average of 17 ± 2.7 vs. 13 ± 3.0, H. armigera to S. litura). The adult lifespan and lifetime fecundity of T. pretiosum were variable but were affected by natal host species and/or host species to which they were exposed. Mean lifespan and fecundity of parasitoids that had developed in H. armigera eggs and were exposed to H. armigera eggs for oviposition were 13.9 ± 1.8 days and 98.7 ± 11.0 adult offspring. By contrast, those that developed in S. litura eggs and were exposed to S. litura eggs for oviposition lived for 7 ± 0.9 days and produced 53.8 ± 8.0 adult offspring. The ovigeny index (OI) was significantly lower in the parasitoids exposed to H. armigera eggs than in those exposed to S. litura eggs, regardless of the natal host, indicating that H. armigera eggs sustain the adult parasitoids better than S. litura eggs. These results are used to predict parasitoid behavior in the field when both hosts are available.     

The collagenous microstructure of cardiac ventricular trabeculae carneae

Cardiac ventricular trabeculae are widely used in the study of cardiac muscle function, primarily because their myocytes are axially-aligned. However, their collagen content has not been rigorously determined. In particular, it is unknown whether the content of collagen differs between specimens originating from the left (LV) and right (RV) ventricles and whether, indeed, either corresponds to the collagen content of the ventricular walls themselves. In order to redress this deficit of knowledge, we have used the techniques of fluorescence confocal microscopy and environmental scanning electron microscopy to quantify the proportion of perimysial collagen comprising the cross-sectional area of trabeculae carneae. In trabeculae from both the RV and LV of adult rat hearts, collagen may occupy as little as 1% or as much as 100% of the cross-section. For specimens of dimensions typically used experimentally, there was no difference in average collagen content (6.03 ± 5.14%, n = 33) of preparations from the two ventricles.     

Chemical modification of corn fiber with ion-exchanging groups

Corn fiber was chemically modified with ion-exchanging groups to prepare water-soluble polysaccharides. The soluble fractions were dialyzed using dialysis tubing (1 kDa) and the material retained inside the tubing was filtered through 10 kDa membranes to separate into fractions with molar mass of 1–10 kDa and greater than 10 kDa. The yield of solubilized material of molar mass higher than 10 kDa (47%) and 1–10 kDa (17%) obtained by sulfonation in the presence of NaOH under vacuum was greater than the yields of the treatment at the ambient pressure (43% and 2%) and also in experiments run with only KOH (40% and 5%) or NaOH (38% and 5%) at ambient pressure. The sugar analysis indicated that they were typical glucuronogalactoarabinoxylans containing 46–57% d-xylose (Xyl), 25–33% l-arabinose (Ara) and 6–12% d-galactose (Gal).     

Effect of molecular size and modification pattern on the internalization of water soluble β-(1 → 3)-(1 → 4)-glucan by primary murine macrophages

It has been shown that β-(1 → 3)-(1 → 4)-glucans (BG34) from barley and oats can trigger recognition and internalization by murine and human macrophages. Increasing evidence has suggested that macrophage recognition and internalization of BG34 are dramatically affected by the purity of BG34, the molecular weight and chemical modification. In this study, we investigated the structural features of BG34 for macrophage recognition and internalization. We prepared homogeneous BG34s of 10 kDa (BG34-10), 200 kDa (BG34-200) and 500 kDa (BG34-500) with high purity, and then introduced green fluorescence FITC to the reducing ends (Re) or main chain (Mc). The results of size exclusion chromatography, ¹³C NMR, fluorescence microscopy, FACS analyses and MTS assay demonstrated that non-toxic BG34 of 10 kDa (BG34-10) effectively trigger macrophage internalization. The internalization was adversely affected by modifying the main chain of BG34-10 but not the reducing end. Studies using blocking antibodies on several CD11b⁺ and CD11b^? cells suggested that CD11b may play an important role in mediating macrophage internalization of BG34-10. Quantitative RT-PCR and intracellular cytokine stain revealed that macrophages generate increased level of CD11b and TNF-α in response to BG34-10. This study for the first time demonstrated the molecular size (10 kDa) and pattern of modification (reducing end modification) for BG34-10 to mediate macrophage internalization. Since BG34 is water soluble, biocompatible and biodegradable FDA-approved material, this mechanism of BG34-10 can be used to design drug delivery system targeting macrophages.     

The effect of limited hydrolysis with Neutrase and ultrafiltration on the anti-adipogenic activity of soy protein

Limited hydrolysis of isolated soy protein (ISP) with Neutrase for 4 h to obtain the hydrolysate (NH4h) revealed the ability to suppress glycerol-3-phosphate dehydrogenase (GPDH) activity and relative lipid accumulation (RLA) in 3T3-L1 cells during differentiation. Lower GPDH activity or RLA indicates higher anti-adipogenic activity. Sequentially fractionating NH4h with 30–1 kDa (kilo-daltons) molecular weight cut-off (MWCO) membranes to obtain the 1 kDa concentrate resulted in further enhancing its anti-adipogenic activity in the cells. The GPDH activity significantly decreased from 280 to 100 U/mg protein (p < 0.05). When comparing the high-performance size-exclusion chromatography (HPSEC) profiles, the most active peptide for the anti-adipogenic activity was primarily composed of peptides with molecular weight between 1300 and 2200 Da. The in vitro effect of gastrointestinal protease on the anti-adipogenic activity of 1 kDa concentrate was also investigated. The results suggested that gastrointestinal proteases have very little effect on anti-adipogenic activity of the concentrate. According to the Western immunoblot analysis, 1 kDa concentrate inhibits adipogenesis by affecting the expression of peroxisome proliferators-activated receptor γ (PPARγ) and the CCAAT/enhancer binding protein α (C/EBPα) during 3T3-L1 cells differentiation.     

Modulation of intracellular chloride channels by ATP and Mg2+

We report the effects of ATP and Mg²⁺ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n = 112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5–2 mmol/l) modulated and/or inhibited the chloride channel activities (n = 38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n = 5/93) or reversible (n = 15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n = 6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5–2 mmol/l) without the presence of Mg²⁺ decreased the chloride channel current (n = 12/14), whereas Mg²⁺ significantly reversed the effect (n = 4/4). We suggest that ATP-intracellular chloride channel interactions and Mg²⁺ modulation of these interactions may regulate different physiological and pathological processes.     

Incubation temperature affects hatching success,embryonic expenditure of energy and hatchling phenotypes of a prolonged egg-retaining snake,Deinagkistrodon acutus (Viperidae)

We used eggs of Deinagkistrodon acutus to study the effects of incubation temperature on hatching success, embryonic expenditure of energy and hatchling phenotypes. One egg from each of the 15 fertile clutches was dissected for determination of egg composition, and a total of 164 eggs were incubated at five constant temperatures. Embryonic mortality increased dramatically at 30 °C, and none of eggs incubated at 32 °C hatched. Within the range from 24 to 30 °C, temperature affected incubation length and most hatchling traits examined. The mean incubation length at 24, 26, 28 and 30 °C was 36.4, 28.7, 21.8 and 15.7 days, respectively. Embryos developing at higher temperatures (28 and 30 °C) consumed more energy but produced less developed (and hence smaller) hatchlings, which characteristically had larger residual yolks but smaller carcasses. A principal component analysis resolved two components (with eigenvalues ⩾1) from ten size (initial egg mass)-free hatchling variables, accounting for 79.3% of variation in the original data. The first component (43.8% variance explained) had high positive loading for size-free values of dry mass, lipid mass, energy contents and ash mass of hatchlings, and the second component (35.5% variance explained) had high positive loading for size-free values of SVL, carcass dry mass and fatbody dry mass. Hatchlings from different incubation temperatures did not differ in scores on the first axis of the principal component analysis, whereas hatchlings from higher incubation temperatures (28 and 30 °C) had significantly lower scores on the second axis than did those from lower incubation temperatures (24 and 26 °C). As the second axis mainly represents traits relating to the developmental condition at hatching, the analysis therefore provided further evidence that eggs incubated at higher temperatures produced less developed hatchlings. Taken together, our data show that the optimal temperatures for embryonic development are relatively low in D. acutus largely due to its use of relatively cool habitats.     

In-depth exploration of Hevea brasiliensis latex proteome and “hidden allergens” via combinatorial peptide ligand libraries

The proteome of Hevea brasiliensis latex has been explored in depth via combinatorial peptide ligand libraries. A total of 300 unique gene products have been identified in this latex, whose proteome has been largely unknown up to the present. In search for unknown allergens, control latex and eluates from the ligand libraries have been fractionated by two-dimensional mapping, blotted and confronted with sera of 18 patients. In addition to the already known and named Hevea major allergens, we have unambiguously detected several others like, for instance: heat shock protein (81 kDa), proteasome subunit (30 kDa), protease inhibitor (8 kDa), hevamine A (43 kDa) and glyceraldehyde-3-phosphate dehydrogenase (37 kDa). Gene Ontology analysis of analyzed fractions has shown that major functions are substantially unchanged after sample treatment, while novel biological functions appeared that were undetectable in the crude sample.     

Efficient oleaginous yeasts for lipid production from lignocellulosic sugars and effects of lignocellulose degradation compounds on growth and lipid production

Microbial lipid production using lignocellulosic biomass is considered an alternative for biodiesel production. In this study, 418 yeast strains were screened to find efficient oleaginous yeasts which accumulated large quantities of lipid when cultivated in lignocellulosic sugars. Preliminary screening by Nile red staining revealed that 142 strains contained many or large lipid bodies. These strains were selected for quantitative analysis of lipid accumulation by shaking flask cultivation in nitrogen-limited medium II containing 70 g/L glucose or xylose or mixture of glucose and xylose in a ratio of 2:1. Rhodosporidium fluviale DMKU-SP314 produced the highest lipid concentration of 7.9 g/L when cultivated in the mixture of glucose and xylose after 9 days of cultivation, which was 55.0% of dry biomass (14.3 g/L). The main composition of fatty acids were oleic acid (40.2%), palmitic acid (25.2%), linoleic acid (17.9%) and stearic acid (11.1%). Moreover, the strain DMKU-SP314 could grow and produce lipid in a medium containing predominantly lignocellulose degradation products, namely, acetic acid, formic acid, furfural, 5-hydroxymethylfurfural (5-HMF) and vanillin, with however, some inhibitory effects. This strain showed high tolerance to acetic acid, 5-HMF and vanillin. Therefore, R. fluviale DMKU-SP314 is a promising strain for lipid production from lignocellulosic hydrolysate.     

Effect of transporting donor or recipient does and their embryos on the outcome of fresh embryo transfer in Boer goats

Between-farm embryo transfer of livestock animals can potentially increase the spread of quality genetic material. However, the transporting of donor or recipient animals or their embryos has become a practical problem. The objective of this study was to compare the effect of transporting donor and recipient does and their embryos between various farms on inter-farm fresh embryo transfer in Boer goats. Results indicate the transportation of donor does within 4 h before embryo collection not to have a significant effect on embryo recovery number, embryo survival rate and the subsequent pregnancy in recipient does. Also, the transportation of embryos at 36.5–38 °C within 2 h before embryo transfer did not significantly affect the embryo survival rate and subsequent pregnancy rate, but the transportation of embryos at 20 °C resulted in a significant (P < 0.05) lower survival rate (41.7%) and pregnancy rate (42.0%). The transportation of recipient does resulted in a significantly lower pregnancy rate (42.0%) and embryo survival rate (32.1%) than the transportation of donor does and embryos. Results suggest the transportation of donor does to be the best method for embryo transfer programs on the farm. Alternatively, the supply of fresh embryos kept at body temperature (36.5 °C) was also preferred for short or long distances between farms.     

Sensitive detection of histamine using fluorescently labeled oxido-reductases

A detection scheme is described by which the histamine contents of biological samples can be established. The scheme is based on the use of methylamine dehydrogenase (MADH) which converts primary amines into the corresponding aldehydes and ammonia. The generated reducing equivalents are subsequently transferred to the physiological partner of MADH, amicyanin, which thereby is converted from the oxidized blue-colored form into the reduced colorless form. The change in absorption is detected by monitoring the fluorescence of a covalently attached Cy5 dye label whose fluorescence is (partly) quenched by Förster resonance energy transfer (FRET) to the Cu-site of the amicyanin. The quenching efficiency and, thereby, the label fluorescence, depends on the oxidation state of the amicyanin. When adding histamine to the assay mixture the proportionality between the substrate concentration and the observed rate of the fluorescence increase has enabled this assay as a sensor method with high sensitivity. The MADH and amicyanin composition can be tuned so that the sensor can be adapted over a broad range of histamine concentrations (13 nM–225 μM). The lowest concentration detected so far is 13 nM of histamine. The sensor retained its linearity up to 225 μM with a coefficient of variation of 11% for 10 measurements of 100 nM histamine in a 100 μL sample volume. The use of a label fluorescing around 660 nm helps circumventing the interference from background fluorescence in biological samples. The sensor has been tested to detect histamine in biological fluids such as fish extracts and blood serum.     

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.     

Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system

Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa granulysin as a secreted protein from insect cells. The 15 kDa granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa granulysin is 0.6 mg/L of insect cell supernatant.