Comber SD et al. (2006) Environ Pollut "Chronic toxicity of sediment-associated linear alkylbenzene sulphonates (LAS) to freshwater benthic organisms."

Webb S, Marshall S, Comber SD, Hoss S, Conrad AU

[Environ Pollut, 2006]

The toxicity of linear alkylbenzene sulphonates (LAS), to freshwater benthic organisms was assessed during exposure to spiked sediment. Lethal and sub-lethal end-points were monitored for two organisms (oligochaete Lumbriculus variegatus and nematode Caenorhabditis elegans). Results demonstrated relatively low toxicity (LOECs >100mg/kg dry weight). No observed effect concentrations (NOECs) of 81mg/kg dw (Lumbriculus) and 100mg/kg dw (Caenorhabditis) were determined. For the oligochaete, no specific endpoint was particularly sensitive to LAS. For the nematode, egg production was the most sensitive endpoint. Significant degradation was measured over the 28-day duration of the Lumbriculus study, equating to a half-life of 20days in sediment.

Sasata RJ et al. (2004) J Biol Chem "Domain swapping localizes the structural determinants of regioselectivity in membrane-bound fatty acid desaturases of Caenorhabditis elegans."

Loewen MC, Sasata RJ, Covello PS, Reed DW

[J Biol Chem, 2004]

Most fatty acid desaturases are members of a large superfamily of integral membrane, O2-dependent iron-containing enzymes that catalyze a variety of oxidative modifications to lipids. Sharing a similar primary structure and membrane topology, these enzymes are broadly categorized according to their positional specificity or regioselectivity, which designates the preferred position for substrate modification. To investigate the structural basis of regioselectivity in membrane-bound desaturases, the Caenorhabditis elegans omega-3 (FAT-1) and delta-12 (FAT-2) desaturases were used as a model system. With the use of unnatural substrates, the regioselectivity of C. elegans FAT-2 was clearly defined as nu+3, i.e., it measures three carbons from an existing double bond. The structural basis for nu+3 and omega-3 regioselectivities was examined through construction and expression of chimeric DNA sequences based on FAT-1 and FAT-2. Each sequence was divided into seven domains, and chimeras were constructed in which specific domains were replaced with sequence from the other desaturase. When tested by expression in yeast using exogenously supplied substrates, chimeric sequences were found in which domain swapping resulted in a change of regioselectivity from nu+3 to omega-3 and vice versa. In this way, the structural determinants of regioselectivity in FAT-1 and FAT-2 have been localized to two interdependent regions: a relatively hydrophobic region between the first two histidine boxes, and the C-terminal region.

Huang Q et al. (2024) Food Chem "A comparative evaluation of the composition and antioxidant activity of free and bound polyphenols in sugarcane tips."

Zhou X, Wen T, Xie C, Li K, Luo Y, Wei T, Huang Q, Huang Z, Lao H, Fang T

[Food Chem, 2024]

The sugarcane tip is abundant in phenolic compounds. Previous studies have concentrated on the effects of free polyphenols, while bound polyphenols were overlooked. In this study, the content of bound polyphenols (SPB) (31.9 ± 0.9 mg GAE/g DW) was significantly higher than free polyphenols (SPF) (3.4 ± 0.1 mg GAE/g DW). A total of 44 free and 31 bound phenolics were identified by the UPLC-EIS-QTOF-MS/MS. Moreover, the antioxidant activity of SPB was more pronounced, as evidenced by its higher ABTS⁺ and DPPH scavenging rates than SPF, which was attributed to the higher tannin content. Furthermore, at all tested concentrations (100 and 200 μg/mL), SPB significantly enhanced the survival and antioxidant enzyme activity of Caenorhabditis elegans (C. elegans), while concurrently reducing ROS levels. High concentrations of SPB even exhibited antioxidant activity comparable to Vitamin C (Vc). The collective findings strongly indicate that SPB holds great potential as an effective antioxidant.

Meesapyodsuk D et al. (2000) Biochemistry "Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase (FAT-1) expressed in Saccharomyces cerevisiae."

Buist PH, Reed DW, Ambrose SJ, Savile CK, Meesapyodsuk D, Covello PS

[Biochemistry, 2000]

To characterize the fatty acid desaturase produced by the fat-1 gene from the nematode Caenorhabditis elegans, the functional expression of this enzyme was effected in the yeast Saccharomyces cerevisiae. The GC-MS analysis of desaturated products derived from various fatty acids, including deuterium-labeled thia fatty acids supplied to growing cultures of transformed yeast, has defined the substrate requirements, regiochemistry, and cryptoregiochemistry of the enzyme. The desaturase acts on substrates of 16-20 carbons with a preference for omega-6 fatty acids, and its regioselectivity was confirmed to be that of an omega-3 desaturase. (omega-x refers to a double bond or desaturation between carbons x and x+1, counting from the methyl end of a fatty acid.) The primary deuterium kinetic isotope effects (KIEs) at C-15 and C-16 of a C18 fatty acid analogue were measured via competitive incubation experiments: While k(H)/k(D) at the omega-3 position was shown to be large (7.8 +/- 0.4), essentially no KIE at the omega-2 position was observed (k(H)/k(D) = 0.99 +/- 0.04). This result indicates that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. The results are discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring differing regioselectivities.

Meesapyodsuk D et al. (2000) Biochem Soc Trans "Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases."

Reed DW, Buist PH, Meesapyodsuk D, Savile CK, Covello PS, Schafer UA, Ambrose SJ

[Biochem Soc Trans, 2000]

In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the omega-3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in baker's yeast (Saccharomyces cerevisiae). Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast. The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16-20 carbons with a preference for omega-6-unsaturated fatty acids. The regioselectivities of both enzymes were confirmed to be that of omega-3 desaturases. The primary deuterium kinetic isotope effects at C-15 and C-16 of a C(18) fatty acid analogue were measured via competitive incubation experiments. Whereas k(H)/k(D) at the omega-3 position was shown to be large, essentially no kinetic isotope effect at the omega-2 position was observed for the plant or the nematode enzymes. These results indicate that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.

Mitsui Y et al. (2017) J Helminthol "Chemoattractant activity of tris-(hydroxymethyl)aminomethane for Brugia pahangi infective third-stage larvae."

Mitsui Y, Urena-Tatis KE, Aoki Y

[J Helminthol, 2017]

Urocanic acid (UCA) is known as a major chemoattractant for Strongyloides stercoralis infective third-stage larvae (L3). Since Brugia pahangi is a skin-penetrating parasitic nematode similar to S. stercoralis, UCA was expected to be a chemoattractant for B. pahangi L3. Thus, the chemoattractant activity of UCA for B. pahangi L3 was assessed. The chemotactic responses of B. pahangi L3 to UCA or acetic acid (CH3COOH) dissolved in amine solutions were assessed using an agar-plate assay. A test solution of 200 mm UCA dissolved in aqueous 270 mm tris(hydroxymethyl)aminomethane (Tris) significantly attracted B. pahangi L3 compared with deionized water (DW), while neither a solution of 200 mm UCA dissolved in aqueous 230 mm ammonia (NH3) nor 290 mm triethylamine (TEA) significantly attracted L3. Similarly, a test solution of 200 mm CH3COOH dissolved with 200 mm Tris significantly attracted L3, but neither a test solution of 200 mm CH3COOH plus 200 mm NH3 nor 200 mm TEA attracted L3. Furthermore, L3 were significantly attracted to 200 mm Tris alone, compared with DW, but avoided 200 mm NH3 and 200 mm TEA. Moreover, the chemoattractant activity of Tris for L3 was observed even at a low concentration of 25 mm, and it was observed in a mild alkaline condition but not in an acidic condition. The present study reveals that Tris is a potential chemoattractant for B. pahangi L3 while UCA is not. This finding will contribute to an understanding of the mechanisms of skin-penetrating infection of filarial L3.

Freire SL et al. (2014) J Vis Exp "Taking advantage of reduced droplet-surface interaction to optimize transport of bioanalytes in digital microfluidics."

Dao S, Thorne N, Wutkowski M, Freire SL

[J Vis Exp, 2014]

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of "lab-on-a-chip" platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique. Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives. Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

Dolfini D et al. (2012) Crit Rev Biochem Mol Biol "NF-Y and the transcriptional activation of CCAAT promoters."

Gatta R, Dolfini D, Mantovani R

[Crit Rev Biochem Mol Biol, 2012]

The CCAAT box promoter element and NF-Y, the transcription factor (TF) that binds to it, were among the first cis-elements and trans-acting factors identified; their interplay is required for transcriptional activation of a sizeable number of eukaryotic genes. NF-Y consists of three evolutionarily conserved subunits: a dimer of NF-YB and NF-YC which closely resembles a histone, and the "innovative" NF-YA. In this review, we will provide an update on the functional and biological features that make NF-Y a fundamental link between chromatin and transcription. The last 25 years have witnessed a spectacular increase in our knowledge of how genes are regulated: from the identification of cis-acting sequences in promoters and enhancers, and the biochemical characterization of the corresponding TFs, to the merging of chromatin studies with the investigation of enzymatic machines that regulate epigenetic states. Originally identified and studied in yeast and mammals, NF-Y - also termed CBF and CP1 - is composed of three subunits, NF-YA, NF-YB and NF-YC. The complex recognizes the CCAAT pentanucleotide and specific flanking nucleotides with high specificity (Dorn et al., 1997; Hatamochi et al., 1988; Hooft van Huijsduijnen et al, 1987; Kim & Sheffery, 1990). A compelling set of bioinformatics studies clarified that the NF-Y preferred binding site is one of the most frequent promoter elements (Suzuki et al., 2001, 2004; Elkon et al., 2003; Marino-Ramirez et al., 2004; FitzGerald et al., 2004; Linhart et al., 2005; Zhu et al., 2005; Lee et al., 2007; Abnizova et al., 2007; Grskovic et al., 2007; Halperin et al., 2009; Hakkinen et al., 2011). The same consensus, as determined by mutagenesis and SELEX studies (Bi et al., 1997), was also retrieved in ChIP-on-chip analysis (Testa et al., 2005; Ceribelli et al., 2006; Ceribelli et al., 2008; Reed et al., 2008). Additional structural features of the CCAAT box - position, orientation, presence of multiple Transcriptional Start Sites - were previously reviewed (Dolfini et al., 2009) and will not be considered in detail here.

Don Fox et al. (2001) International C. elegans Meeting "Securin and"

Andy Golden, Barry Chestnut, Don Fox

[International C. elegans Meeting, 2001]

Cell division results in the precise halving of genetic material. In yeast, such partitioning requires the protease separin 1 . This protease acts at the metaphase to anaphase transition to degrade sister chromatid cohesion, which in turn permits chromosomes to segregate to opposite poles of a dividing cell . In addition to facilitating chromosome segregation, separin promotes spindle elongation in S. cerevisiae through the activity of a calcium-binding domain 2 . Separin activity also depends heavily on the anaphase inhibitor securin, which localizes separin to both the nucleus and spindle mid-zone, and inhibits its protease activity until anaphase. Here, we report a requirement for the C. elegans homologue of yeast separin during oocyte meiosis. RNAi of this homologue, which contains both the protease and calcium binding domains of S. cerevisiae separin, results in multi-nucleated one-cell embryos with multiple spindles. In these embryos, the chromosomes are disorganized and the embryos fail to produce polar bodies. Taken together, these results suggest a role for C.elegans separin in chromosome segregation and cytokinesis in the early embryo. Currently, we are investigating if C. elegans separin also plays a role in exit from M-phase of the cell cycle. Additionally, we hope to identify protein partners of C.elegans separin by using the protein as bait in a yeast 2-hybrid screen. This screen may prove the most effective way of identifying a C. elegans securin homologue, as all of the known securins from other organisms demonstrate no amino acid conservation. Also, to date, no separin interacting proteins other than securin have been identified. Results of the screen will be discussed at the meeting. 1.Uhlman, F., Wernic, D., Poupart, M., Koonin, E., and K. Nasmyth. 2000. Cleavage of Cohesin by the CD Clan Protease Separin Triggers Anaphase in Yeast. Cell 103:375-386. 2.Jensen, S., Segal, M., Clarke, D., and S. Reed. 2001. A Novel Role of the Budding Yeast Separin Esp1 in Anaphase Spindle Elongation: Evidence that Proper Spindle Associatioin of Esp1 is Regulated by Pds1. J.Cell Biol. 152:27-40.

Tanaka YU et al. (1996) Worm Breeder's Gazette "Tissue-specific expression and RNA binding specificity of the C. elegans homologue of the spliceosome-associated protein SAP 49."

Sakamoto H, Tanaka YU, Ohta A

[Worm Breeder's Gazette, 1996]

We have isolated a C. elegans cDNA (accession no. U24189) that encodes a putative 388 amino-acid RNA-binding protein with two RNA recognition motifs (RRMs). The RNA-binding protein shows extensive similarity to the spliceosome-associated protein 49 (SAP 49) which has been shown to be tightly involved in U2 snRNP function in mammalian cells (Champion-Arnaud and Reed, Genes Dev. 8, 1974-83, 1994). Surprisingly, the cDNA also contains two additional ORFs upstream of the coding region for the C. elegans SAP 49 homologue (cSAP49). The product deduced from the first ORF shows no homology with any known protein, whereas the product of the second ORF encodes an actin-related protein. Since very short spacer regions containing the possible poly(A) and 3' splice sites are present between these ORFs, and since these ORFs are continuous in the genome sequence (CEC08B11; accession no. Z46676), it is suggested that these ORFs are first transcribed in a single primary transcript and then processed into three independent mRNAs by trans-splicing mechanism. Indeed, we confirmed three different mRNAs corresponding to the three putative proteins by Northernblot analysis. By microinjection of the GFP- and LacZ-fusion constructs of cSAP49 under the control of the native promoter, we have found that it is expressed at the restricted regions of the developing worm, mainly in the nuclei of intestine tissues. This expression pattern is puzzling because the human SAP 49 is thought to bear the essential function in all cells. In addition, using an in vitro selection method, we have demonstrated that cSAP49 possesses specific RNA binding ability which is attributed to the second RRM. Its RNA binding is in essentially a sequence-specific manner but is affected by the surrounding sequence context, suggesting that cSAP49 may recognize the secondary or tertiary structure as an additive information. We speculate that a possible target for the cSAP49 may be the U2 snRNA since it is highly structured and contains a sequence similar to a part of the consensus sequence of cSAP49-selected RNAs. We are currently examining whether cSAP49 really binds to the U2 snRNA in the presence or absence of another U2-associated protein SAP 145, and whether there would be other tissue-specific SAP 49 homologue in C. elegans.