Biomarker responses in New Zealand green-lipped mussels Perna canaliculus exposed to microplastics and triclosan.

Microplastics (MPs) are of increasing concern for filter feeding marine and freshwater species. Additionally MPs can sorb hydrophobic contaminants from the water, potentially providing an additional pathway of exposure of aquatic species to contaminants. An acute 48 h laboratory study was conducted to investigate the effects of microplastics and triclosan, both individually and combined, on New Zealand's green-lipped mussel, Perna canaliculus. Biomarkers included clearance rate, oxygen uptake, byssus production; and superoxide dismutase (SOD) activity, glutathione-S-transferase (GST) activity and lipid peroxidation in the gill tissue. Microplastics and triclosan, both individually and combined significantly decreased oxygen uptake and byssus production. These physiological responses were not observed when the microplastics were spiked with triclosan. Triclosan, both alone and spiked to microplastics, increased mussel oxidative stress markers including SOD activity and lipid peroxidation. An enhanced effect was observed on the SOD enzyme activity when mussels were exposed to microplastics spiked with triclosan. No effects on the biochemical biomarkers were observed for mussels exposed to microplastic only. Microplastics enhanced the uptake of triclosan in mussel tissue compared with triclosan only treatments indicating that microplastics potentially provide an additional pathway of exposure to hydrophobic contaminants.

ADF/Cofilin-actin rods in neurodegenerative diseases.

Dephosphorylation (activation) of cofilin, an actin binding protein, is stimulated by initiators of neuronal dysfunction and degeneration including oxidative stress, excitotoxic glutamate, ischemia, and soluble forms of beta-amyloid peptide (Abeta). Hyperactive cofilin forms rod-shaped cofilin-saturated actin filament bundles (rods). Other proteins are recruited to rods but are not necessary for rod formation. Neuronal cytoplasmic rods accumulate within neurites where they disrupt synaptic function and are a likely cause of synaptic loss without neuronal loss, as occurs early in dementias. Different rod-inducing stimuli target distinct neuronal populations within the hippocampus. Rods form rapidly, often in tandem arrays, in response to stress. They accumulate phosphorylated tau that immunostains for epitopes present in "striated neuropil threads," characteristic of tau pathology in Alzheimer disease (AD) brain. Thus, rods might aid in further tau modifications or assembly into paired helical filaments, the major component of neurofibrillary tangles (NFTs). Rods can occlude neurites and block vesicle transport. Some rod-inducing treatments cause an increase in secreted Abeta. Thus rods may mediate the loss of synapses, production of excess Abeta, and formation of NFTs, all of the pathological hallmarks of AD. Cofilin-actin rods also form within the nucleus of heat-shocked neurons and are cleared from cells expressing wild type huntingtin protein but not in cells expressing mutant or silenced huntingtin, suggesting a role for nuclear rods in Huntington disease (HD). As an early event in the neurodegenerative cascade, rod formation is an ideal target for therapeutic intervention that might be useful in treatment of many different neurological diseases.

Haemolymph glucose concentrations of juvenile rock lobsters, Jasus edwardsii, feeding on different carbohydrate diets.

Postprandial changes in haemolymph glucose concentration ([Glc]H) were measured in 4-day-fasted juvenile intermoult spiny lobsters, Jasus edwardsii, provided with meals composed of glycogen, maltose, sucrose, glucose, or fructose in a gelatine base, or with gels of the algal glycans agar, alginate and carrageenan. Baseline [Glc]H was 0.61+/-0.02 mmol L(-1). After consumption of glycogen, maltose or sucrose, [Glc]H approximately doubled, peaked after 3 h and returned to baseline between 12 and 24 h. Glucose and fructose meals were followed by periods of sustained hyperglycaemia lasting more than 24 h (peaking at approximately 2.5 times baseline at 6 and 3 h respectively). Suggested explanations for augmented hyperglycaemic responses to glucose and fructose are: 1) these monosaccharides by-passed contact digestion and absorption in the R-cells of the digestive gland, directing them away from storage and toward transepithelial scavenging routes; or 2) glucose and fructose directly elicited release of crustacean hyperglycaemic hormone via a chemosensory reflex. Agar and alginate induced significant postprandial glycaemic responses, consistent with reports of carbohydrases in this species and indicating their potential for inclusion in artificial diets as both binders and energy sources. Carrageenan, a highly sulphated galactan, did not produce a glycaemic response. The measurement of glycaemic responses is a quick method of obtaining nutritional information on carbohydrates considered for inclusion in formulated diets prior to lengthy growth trials.

Dynamic DNA contacts observed in the NMR structure of winged helix protein-DNA complex.

Genesis is an HNF-3/fkh homologous protein. By using multi-dimensional NMR techniques, we have obtained the solution structure and backbone dynamics of Genesis complexed with a 17 base-pair DNA. Our results indicate that both the local folding and dynamic properties of Genesis are perturbed when it binds to the DNA site. Our data show that a conserved flexible amino acid sequence (wing 1) makes dynamic contacts to DNA in the complex and a short helix is induced by Genesis-DNA interactions. Our data indicate that, unlike the HNF-3gamma/DNA complex, a magnesium ion is not required in forming the stable Genesis-DNA complex.

Structural changes in the region directly adjacent to the DNA-binding helix highlight a possible mechanism to explain the observed changes in the sequence-specific binding of winged helix proteins.

The hepatocyte nuclear factor 3 (HNF-3)/fork head (fkh) family contains a large number of transcription factors and folds into a winged helix motif. Despite having almost invariable amino acid sequences in their principal DNA-binding helices, HNF-3/fkh proteins show a wide diversity of sequence-specific binding. Previous studies of chimeric HNF-3/fkh proteins demonstrated that the binding specificity is primarily influenced by a region directly adjacent to the binding helix. We report our findings of an NMR structural study performed on an HNF-3/fkh family member (Genesis, formerly HFH-2) and compare it to that of another family member (HNF-3gamma) complexed to DNA and determined by X-ray crystallography. It is found that in comparison to HNF-3gamma, Genesis contains an extra small helix directly prior to the N terminus of the primary DNA contact helix. Due to the insertion of this helix, a shorter and slightly re-positioned primary DNA contact helix is observed, which we believe leads to the DNA-binding specificity differences among family members.

Sequence specific collective motions in a winged helix DNA binding domain detected by 15N relaxation NMR.

The recognition between transcription factors and their DNA binding sites is a highly dynamic process. During transcriptional regulation, transcription factors must bind to or dissociate from their cognate DNA binding sites. The winged helix DNA binding motif is one of many highly conserved DNA binding motifs identified in transcription factors. Backbone dynamics has been studied on the 15N- and 2H-enriched winged helix family member Genesis. Our data show that the overall motions of the single domain Genesis are better described by more than two autocorrelation times (taum). Our data also demonstrate that Genesis shows structure specific conformation exchange characterized by Rex. Therefore, our results indicate that the structure of Genesis is highly dynamic and that secondary structure elements in Genesis have collective motions in the nanosecond to millisecond time scale. Since the winged helix DNA binding motif is highly conserved, this unique dynamic property observed in Genesis is also likely to be conserved in other winged helix family members and important in DNA binding.

Evidence that the DNA binding specificity of winged helix proteins is mediated by a structural change in the amino acid sequence adjacent to the principal DNA binding helix.

We present the first structural evidence supporting the hypothesis that the binding specificity of the winged helix DNA binding motif is mediated by residues adjacent to the alpha-helix (H3), the moiety which is primarily involved in the interaction with DNA. Using NMR to determine secondary structural elements of a winged helix family member, Genesis (formerly HFH-2), and comparing these with those found in the X-ray crystal structure of the HNF-3gamma/DNA complex [Clark, K. L., Halay, E. D., Lai, E., & Burley, S. K. (1993) Nature 364, 412-420], we show that the major differences observed occur for H3 and the region immediately prior to this. H3 in Genesis is slightly shorter than in HNF-3gamma and, in addition, we observe an extra small helix (H4) in the region between H2 and H3 which is not found in the HNF-3gamma/DNA complex. This is significant as it has been shown previously [Overdier, D. G., Porcella, A., & Costa R. H. (1994) Mol. Cell. Biol. 14, 2755-2766] that the DNA-binding specificity is influenced by amino acid residues in this region.

An anti-neoplastic glycan isolated from Mycobacterium bovis (BCG vaccine).

Tice substrain BCG is used clinically as an immunotherapeutic agent against superficial bladder cancer. A boiling-water extract of this BCG showed anti-tumour activity against a murine S180 sarcoma model and was fractionated into three fractions, A, B and C, by the use of Sephadex LH-20 chromatography. An anti-tumour glucan, PS1A1, was isolated from fraction PS1A with Sephadex G-75. The molecular mass of PS1A1 was between 65 and 87 kDa by Sephadex G-100 chromatography. The structure of PS1A1 was investigated by one- and two-dimensional NMR spectroscopy and methylation analysis and was demonstrated to be primarily 1-->6-alpha-linked glucose units. We postulate that the repeating unit is: [Formula: see text]

Polarimetry and 13C n.m.r. show that the hydrolyses of beta-D-glucopyranosyl fluoride by beta(1-->3)-glucanases from Phanerochaete chrysosporium and Sporotrichum dimorphosporum have opposite stereochemistries.

The time courses of optical rotation and fluoride ion release during hydrolysis of beta-D-glucopyranosyl fluoride by the beta(1-->3)-glucanase of Phanerochaete chrysosporium (J. L. Copa-Patiño and P. Broda, unpublished work) indicated that the initial sugar product was beta-D-glucopyranose. This was confirmed by monitoring the hydrolysis of 1-[13C]beta-D-glucopyranosyl fluoride by this enzyme with 13C n.m.r. (without proton decoupling). The same two techniques were used to confirm that hydrolysis of beta-D-glucopyranosyl fluoride by the exo beta(-->3)-glucanase of 'Basidiomycete QM 806' (identified as Sporotrichum dimorphosporum) yielded alpha-glucopyranose as first sugar product, in accordance with previous results using laminarin as substrate [Parrish and Reese (1963) Carbohydr. Res. 3, 424-429; Nelson (1970) J. Biol. Chem. 245, 869-872].

Hydrolyses of alpha- and beta-cellobiosyl fluorides by cellobiohydrolases of Trichoderma reesei.

Cellobiohydrolase II hydrolyses alpha- and beta-D-cellobiosyl fluorides to alpha-cellobiose at comparable rates, according to Michaelis-Menten kinetics. The stereochemistry, absence of transfer products and strict hyperbolic kinetics of the hydrolysis of alpha-cellobiosyl fluoride suggest that the mechanism for the alpha-fluoride may be the enzymic counterpart of the SNi reaction observed in the trifluoroethanolysis of alpha-glucopyranosyl fluoride [Sinnott and Jencks (1980) J. Am. Chem. Soc. 102, 2026-2032]. The absolute factors by which this enzyme accelerates fluoride ion release are small and greater for the alpha-fluoride than for the beta, suggesting that its biological function may not be just glycoside hydrolysis. Cellobiohydrolase I hydrolyses only beta-cellobiosyl fluoride, which is, however, an approx. 1-3% contaminant in alpha-cellobiosyl fluoride as prepared and purified by conventional methods. Instrumental assays for the various components of the cellulase complex are discussed.