The crystal structure of Echinococcus granulosus fatty-acid-binding protein 1

We describe the 1.6 Å crystal structure of the fatty-acid-binding protein EgFABP1 from the parasitic platyhelminth Echinococcus granulosus. E. granulosus causes hydatid disease, which is a major zoonosis. EgFABP1 has been implicated in the acquisition, storage, and transport of lipids, and may be important to the organism since it is incapable of synthesising most of its lipids de novo. Moreover, EgFABP1 is a promising candidate for a vaccine against hydatid disease.The crystal structure reveals that EgFABP1 has the expected 10-stranded β-barrel fold typical of the family of intracellular lipid-binding proteins, and that it is structurally most similar to P2 myelin protein. We describe the comparison of the crystal structure of EgFABP1 with these proteins and with an older homology model for EgFABP1.The electron density reveals the presence of a bound ligand inside the cavity, which we have interpreted as palmitic acid. The carboxylate group of the fatty acid interacts with the protein's P2 motif, consisting of a conserved triad R…R-x-Y. The hydrophobic tail of the ligand assumes a fairly flat, U-shaped conformation and has relatively few interactions with the protein.We discuss some of the structural implications of the crystal structure of EgFABP1 for related platyhelminthic FABPs.     

Holocarboxylase synthetase regulates expression of biotin transporters by chromatin remodeling events at the SMVT locus

The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. Jurkat lymphoma cells were cultured in media containing 0.025, 0.25, or 10 nmol/l biotin. The nuclear translocation of HCS correlated with biotin concentrations in media; the relative enrichment of both HCS and K12Bio H4 at SMVT promoter 1 (but not promoter 2) increased by 91% in cells cultured in medium containing 10 nmol/l biotin compared with 0.25 nmol/l biotin. This increase of K12Bio H4 at the SMVT promoter decreased SMVT expression by up to 86%. Biotin homeostasis by HCS-dependent chromatin remodeling at the SMVT promoter 1 locus was disrupted in HCS knockdown cells, as evidenced by abnormal chromatin structure (K12Bio H4 abundance) and increased SMVT expression. The findings from this study are consistent with the theory that HCS senses biotin, and that biotin regulates its own cellular uptake by participating in HCS-dependent chromatin remodeling events at the SMVT promoter 1 locus in Jurkat cells.     

Identification and tissue-specific distribution of sulfated glycosaminoglycans in the blood-sucking bug Rhodnius prolixus (Linnaeus)

We have previously characterized heparan sulfate (HS) as the major ovarian sulfated glycosaminoglycan (GAG) in females of Rhodnius prolixus, while chondroitin sulfate (CS) was the minor component. Using histochemical procedures we found that GAGs were concentrated in the ovarian tissue but not found inside the oocytes. Here, we extend our initial observations of GAG expression in R. prolixus by characterizing these molecules in other organs: the fat body, intestinal tract, and the reproductive tracts. Only HS and CS were found in the three organs analyzed, however CS was the major GAG species in these tissues. We also determined the compartmental distribution of GAGs in these organs by histochemical analysis using 1,9-dimethylmethylene blue, and evaluated the specific distribution of CS within both male and female reproductive tracts by immunohistochemistry using an anti-CS antibody. We also determined the GAG composition in eggs at days 0 and 6 of embryonic development. Only HS and CS were found in eggs at day 6, while no sulfated GAGs were detected at day 0. Our results demonstrate that HS and CS are the only sulfated GAG species expressed in the fat body and in the intestinal and reproductive tracts of Rhodnius male and female adults. Both sulfated GAGs were also identified in Rhodnius embryos. Altogether, these results show no qualitative differences in the sulfated GAG composition regarding tissue-specific or development-specific distribution.     

Epidemiology and Genetic Epidemiology of the Liver Function Test Proteins

Background

The liver function test (LFT) is among the most commonly used clinical investigations to assess hepatic function, severity of liver diseases and the effect of therapies, as well as to detect drug-induced liver injury (DILI).

Aims

To determine the relative contribution of genetic and environmental factors as well as test and quantify the effects of sex, age, BMI and alcohol consumption to variation in liver function test proteins - including alanine amino transaminase (ALT), Albumin, gamma glutamyl transpeptidase (GGT), total bilirubin, total protein, total globulin, aspartate transaminase (AST), and alkaline phosphotase (ALP) - using the classical twin model.

Methods

Blood samples were collected from a total of 5380 twin pairs from the TwinsUK registry. We measured the expression levels of major proteins associated with the LFT, calculated BMI from measured weight and height and questionnaires were completed for alcohol consumption by the twins. The relative contribution of genetic and environmental factors to variation in the LFT proteins was assessed and quantified using a variance components model fitting approach.

Results

Our results show that (1) variation in all the LFTs has a significant heritable basis (h² ranging from 20% to 77%); (2) other than GGT, the LFTs are all affected to some extent by common environmental factors (c² ranging from 24% to 54%); and (3) a small but significant proportion of the variation in the LFTs was due to confounding effects of age, sex, BMI, and alcohol use.

Conclusions

Variation in the LFT proteins is under significant genetic and common environmental control although sex, alcohol use, age and BMI also contribute significantly to inter-individual variation in the LFT proteins. Understanding the underlying genetic contribution of liver function tests may help the interpretation of their results and explain wide variation among individuals.     

Basic amino acid residues in the beta-structure region contribute, but not critically, to presynaptic neurotoxicity of ammodytoxin A

The molecular mechanism of action of presynaptically toxic secreted phospholipases A2 (sPLA2s) isolated from snake venoms is not completely understood. It has been proposed that the positive charge in the beta-structure region is important for their toxic activity. To test this hypothesis, we characterised several mutants of ammodytoxin A (AtxA) possessing substitution of all five basic residues in this region. The mutations had relatively little influence on the catalytic activity of AtxA, either on charge-neutral or anionic phospholipid vesicles. An exception was R72 when replaced by a hydrophobic (higher activity) or an acidic (lower activity) residue. Lethal potencies of the eight single site mutants were up to four times lower than that of the wild-type, whereas the triple mutant (K74S/H76S/R77L) was 13-fold less toxic. The substitutions also lowered the affinity of the toxin, slightly to moderately, for the neuronal receptors R25 and R180. Interaction with calmodulin was only slightly affected by substitutions of K86, more by those of the K74/H76/R77 cluster and most by those of R72 (up to 11-fold lower binding affinity). The results clearly indicate that the basic amino acid residues in the beta-region of AtxA contribute to, but are not necessary for, its neurotoxic effect.     

Oxidative stress and antioxidant content in Chlorella vulgaris after exposure to ultraviolet-B radiation

Growth of Chlorella vulgaris was measured in cultures irradiated with 0, 0.8, 2.0 and 4.4 kJ m² UV-B. Growth expressed as chlorophyll content, declined significantly with increased UV-B dose. Ultraviolet-B irradiated cultures in log phase of growth showed a 284% increase in oxygen radical generation and a 145% increase in lipid peroxidation compared with unirradiated cultures, whereas cultures in the stationary growth phase showed no significant changes in these parameters. The activities of superoxide dismutase and catalase increased by 40 and 500%, respectively, after exposure to a UV-B dose of 4.4 kJ m⁻². Contents of the lipophilic antioxidants α-tocopherol and β-carotene increased by 180 and 63 amol cell⁻¹ respectively, between log and stationary phases in unirradiated cultures; but in UV-B-irradiated cultures these increases were significantly depressed. Photoreducing capacities of chloroplasts were decreased following UV-B irradiation of both isolated chloroplasts and those isolated from irradiated algae. Cells exposed to UV-B exhibited increased size and starch accumulation. These results suggest that oxidative stress conditions related to UV-B exposure trigger an antioxidant response that includes an increase in the activity of the antioxidant enzymes (superoxide dismutase and catalase).     

Limnology of Crater Lakes in Los Tuxtlas,Mexico

Investigations on the abundance, biomass and position of heterotrophic flagellates (HF) in the benthic microbial food web of a melt water stream on King George Island, Antarctic Peninsula, were undertaken during the Antarctic summer from 23rd December 1997 until 13th March 1998. Abundance and biomass of potential HF resources (picophotoautotrophic and non-photoautotrophic bacteria) as well as potential predators on HF (ciliates and meiofauna) were also investigated. HF abundance ranged from approximately 9 × 10³ to 81 × 10³ cells cm^–3, values which fall into the same range as those found in lower latitudes. Numerically important benthic HF were euglenids, kinetoplastids, thaumatomastigids and especially chrysomonads. Most species identified have been shown to have a worldwide distribution. Abundance of the benthic ciliates ranged from 27 to 950 cells cm^–3. Mean bacterial abundance was 1.9 × 10⁷ and 5.2 × 10⁸ cells cm^–3 for picophotoautotrophic and non-photoautotrophic benthos, respectively. The well-developed microbial community was able to support the large number of nematods, gastotrichs, tardigrads and rotifers with abundances reaching more than 1000 individuals cm^–3. The largest portion of heterotrophic biomass was formed by the meiofauna with a mean of 63 g C cm^–3, followed by that of the heterotrophic bacteria with 4.80 g C cm^–3. Picophotoautotrophic bacteria contributed a mean of 1.37 g C cm^–3. HF and ciliates mean biomass was 0.61 and 1.99 g C cm^–3, respectively, with the HF biomass comprising between <10 and 70% of the total protozoan biomass. The data obtained in this study identify the melt water stream as a hot-spot of heterotrophic microbial and meiofaunal activity during the austral summer. The HF in the melt water stream formed a diverse group in terms of taxa and potential feeding types. Chrysomonads, kinetoplastids, euglenids and thaumatomastigida were the most abundant taxa. A classification into feeding types identified an average of 34% of the total HF as bacterivorous while all others were able to utilise other, larger organisms as resources. Potential trophic interactions between HF and bacteria and higher trophic levels are discussed.     

Short-term analysis of the phytoplankton structure and dynamics in two ponds with distinct trophic states from Cierva Point (maritime Antarctica)

Phytoplankton communities dominating Musgos and Papúa ponds with differing trophic states were sampled over 3 days enabling the detection of the physiological and population responses of microalgae to short-scale changes in biotic and abiotic factors, rather than frequently analyzed changes in community composition responses to long-scale environmental changes. We hypothesized that both environments undergoing diel changes would be dominated by phytoplankton with generalist strategies, while community structure would be mostly dictated by the trophic state of each water body. The phytoplankton biovolumes of both ponds were strongly dominated by euplanktonic nanoflagellated Chlorophyta, while phycocyanin-rich picocyanobacteria dominated the picophytoplankton. Parallel diel cycles of air and water temperatures were more pronounced on a sunny, warm day which prompted algal photosynthesis, revealed by strong increases in dissolved oxygen and pH. Nutrient and phytoplanktonic chlorophyll a confirmed the hypertrophic condition of Papúa pond. This accounted for the distinct community composition encountered in each pond, which remained stable throughout the study, as revealed by the SIMI index. The inverse relationship between the chl a/abundance ratio and the abundances of dominant species together with varying net growth rates (k′) showed algal reproduction, yet densities remained rather stable in both cases. In Musgos pond, fluctuations in k′ for small and median ciliates shadowed those of pico- and nanophytoplankton, respectively, strongly suggesting that they can control algal growth in these 2-level trophic chains.