Egg Case Protein 3: A Constituent of Black Widow Spider Tubuliform Silk.

Spider silk has outstanding mechanical properties, rivaling some of the best materials on the planet. Biochemical analyses of tubuliform silk have led to the identification of TuSp1, egg case protein 1, and egg case protein 2. TuSp1 belongs to the spidroin superfamily, containing a non-repetitive N- and C-terminal domain and internal block repeats. ECP1 and ECP2, which lack internal block repeats and sequence similarities to the highly conserved N- and C-terminal domains of spidroins, have cysteine-rich N-terminal domains. In this study, we performed an in-depth proteomic analysis of tubuliform glands, spinning dope, and egg sacs, which led to the identification of a novel molecular constituent of black widow tubuliform silk, referred to as egg case protein 3 or ECP3. Analysis of the translated ECP3 cDNA predicts a low molecular weight protein of 11.8 kDa. Real-time reverse transcription-quantitative PCR analysis performed with different silk-producing glands revealed ECP3 mRNA is predominantly expressed within tubuliform glands of spiders. Taken together, these findings reveal a novel protein that is secreted into black widow spider tubuliform silk.

The effect of Artichoke (Cynara scolymus L.) on the expression of calcium-binding proteins in the eggshell gland of laying hens.

The purpose of the present work is to investigate the effect of dietary-supplemented artichoke (Cynara scolymus L.) on the mRNA expression of calbindin 1 (Calb1), osteopontin (Spp1), albumin (Alb) and CALB1 protein in the eggshell gland (ESG) of laying hens. A total of 80 ISA Brown hens (each at 40 weeks of age) were randomly divided into two groups: a control and a treated group. All poultry received 130 g/day of compound feed for laying hens but the treated hens' diet was also supplemented with 3g/kg of dried and milled artichoke (Cynara scolymus L.). The increase of the Ca content in blood of the treated hens was established. Significantly decrease of Spp1 mRNA transcripts was found in the eggshell gland of the treated hens, while the mRNA level of Alb was increased. The relative expression of Calb1 mRNA tended to increase in the treated group. The expression of calbindin protein in the cytoplasm of glandular cells of the shell gland was defined by immunohistochemical method. Very strong signals of calbindin were observed in the treated group. The supplementation of the laying hens' diet with dried artichoke (C. scolymus L.) led to a significant increase of Ca content in blood that was reflected in the changes of expression of the eggshell gland genes involved in the mineralization of eggshell.

Transcriptome and Parasitome Analysis of Beet Cyst Nematode Heterodera schachtii.

Beet cyst nematodes depend on a set of secretory proteins (effectors) for the induction and maintenance of their syncytial feeding sites in plant roots. In order to understand the relationship between the beet cyst nematode H. schachtii and its host, identification of H. schachtii effectors is crucial and to this end, we sequenced a whole animal pre-infective J2-stage transcriptome in addition to pre- and post-infective J2 gland cell transcriptome using Next Generation Sequencing (NGS) and identified a subset of sequences representing putative effectors. Comparison between the transcriptome of H. schachtii and previously reported related cyst nematodes and root-knot nematodes revealed a subset of esophageal gland related sequences and putative effectors in common across the tested species. Structural and functional annotation of H. schachtii transcriptome led to the identification of nearly 200 putative effectors. Six putative effector expressions were quantified using qPCR and three of them were functionally analyzed using RNAi. Phenotyping of the RNAi nematodes indicated that all tested genes decrease the level of nematodes pathogenicity and/or the average female size, thereby regulating cyst nematode parasitism. These discoveries contribute to further understanding of the cyst nematode parasitism.

Effect of lithium chloride on d-galactose induced organs injury: Possible antioxidative role.

The aging process is concerned with oxidative stress and causing malfunction of various organs such as the liver, kidney and heart. Lithium (Li) salts have shown anti-manic, anti-suicidal, and antioxidant properties. The current study is aimed to evaluate the possible inhibitory effects of various doses (10, 20 & 40mg/ml/kg) of Lithium chloride (LiCl) on D-galactose (D-gal)-produced aging model and explore the underlying mechanism. In the study 40 male rats were randomly alienated into 8 groups i.e. saline, LiCl (10, 20 & 40mg/ml/kg), D-gal and D-gal+LiCl (10, 20 & 40 mg/ml/kg). D-gal was given at a dosage of 300mg/ml/kg$ and animals received their respective treatment for 6 weeks [intraperitoneally (I.P), once daily]. After 2 weeks animals were decapitated and organs (liver, kidney, and heart) were removed for antioxidant assays. Blood was also collected for biochemical parameters. LiCl substantially decreased oxidative strain marker and increased enzymatic antioxidants in the liver, kidney, and heart of D-gal treated rats. LiCl also decreased serum alanine aminotransferase (ALT), aspartate transaminase (AST), creatine, urea, CK-MB, triglyceride, cholesterol, low-density lipoprotein (LDL) and increased high-density lipoprotein (HDL) in D-gal treated animals. High dose (80mg/ml/kg) of LiCl observed as the most effective dose against D-gal induced alterations. These finding LiCl inhibits D-gal induced liver, kidney and heart damages via its antioxidant potential.

Fluorescent nanoparticles in the popular pizza: properties, biodistribution and cytotoxicity.

Food-borne nanoparticles that are generated during the thermal processing of various consumed foods are of great concern due to their unique properties. In this study, the presence of fluorescent nanoparticles (FNPs) in pizza, their biodistribution and cytotoxicity were investigated. The spherical FNPs have a diameter of about 3.33 nm. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy analysis revealed that they contained 68.21% C, 27.44% O, 2.75% N and 1.60% S, and the functional groups on their surface included -OH, -COOH, C[double bond, length as m-dash]C, -NH2 and C[double bond, length as m-dash]O. In vitro and in vivo biodistribution of pizza FNPs was evaluated using normal rat kidney (NRK) cells, onion epidermal cells, Caenorhabditis elegans and mice. The fluorescence microscopy images clearly indicate that the pizza FNPs appear to be localized within the cytoplasm. However, the FNPs remained restricted to the extracellular space of the onion epithelium and did not enter the onion cell cytoplasm because of the cell wall. The FNPs were swallowed by the Caenorhabditis elegans worms when exposed to food OP50 and distributed within the pharynx, intestine and anus. Obvious fluorescence of the FNPs in the stomach, intestine, liver, lung and kidney was observed for the FNPs in mouse organs, but not the brain, heart, and spleen. Furthermore, the produced FNPs were found to cause cell cycle arrest at the G0/G1 phase in NRK cells, and resulted in cell apoptosis at high doses. The outcome of this research offers an important insight into the nature of thermal processing-induced nanoparticles and their in vivo and in vitro biological effects.

Anthocyanin Tissue Bioavailability in Animals: Possible Implications for Human Health. A Systematic Review.

Anthocyanins (ACNs) are promising health-enhancing phenolic compounds. We focus on ACN animal tissue bioavailability to provide an evidentiary link between tissue ACNs and their associated health properties. We performed a systematic review of electronic libraries; 279 results were retrieved, and 13 publications met inclusion criteria. Extracted information included animal model employed, administration route, doses, analysis method, and ACN concentration values in tissues. Total ACN concentrations were detected in mice kidney (2.17 × 105 pmol/g), liver (1.73 × 105 pmol/g), heart (3.6 × 103 pmol/g), and lung (1.16 × 105 pmol/g); and in pig brain (6.08 × 103 pmol/g). ACNs showed a predominance of parent ACNs in long-term experiments versus an ACN metabolite predominance in short-term experiments. ACNs detected in animal tissues, such as cyanidin-3-glucoside, suggest it may have an important role in human health. This information could be useful to determine proper ACN-intake biomarkers in biological samples in futures studies.

Dynamics of actinotrichia regeneration in the adult zebrafish fin.

The skeleton of adult zebrafish fins comprises lepidotrichia, which are dermal bones of the rays, and actinotrichia, which are non-mineralized spicules at the distal margin of the appendage. Little is known about the regenerative dynamics of the actinotrichia-specific structural proteins called Actinodins. Here, we used immunofluorescence analysis to determine the contribution of two paralogous Actinodin proteins, And1/2, in regenerating fins. Both proteins were detected in the secretory organelles in the mesenchymal cells of the blastema, but only And1 was detected in the epithelial cells of the wound epithelium. The analysis of whole mount fins throughout the entire regenerative process and longitudinal sections revealed that And1-positive fibers are complementary to the lepidotrichia. The analysis of another longfin fish, a gain-of-function mutation in the potassium channel kcnk5b, revealed that the long-fin phenotype is associated with an extended size of actinotrichia during homeostasis and regeneration. Finally, we investigated the role of several signaling pathways in actinotrichia formation and maintenance. This revealed that the pulse-inhibition of either TGFß/Activin-ßA or FGF are sufficient to impair deposition of Actinodin during regeneration. Thus, the dynamic turnover of Actinodin during fin regeneration is regulated by multiple factors, including the osteoblasts, growth rate in a potassium channel mutant, and instructive signaling networks between the epithelium and the blastema of the regenerating fin.

Global Transcriptome Analysis of the Tentacle of the Jellyfish Cyanea capillata Using Deep Sequencing and Expressed Sequence Tags: Insight into the Toxin- and Degenerative Disease-Related Transcripts.

BACKGROUND: Jellyfish contain diverse toxins and other bioactive components. However, large-scale identification of novel toxins and bioactive components from jellyfish has been hampered by the low efficiency of traditional isolation and purification methods. RESULTS: We performed de novo transcriptome sequencing of the tentacle tissue of the jellyfish Cyanea capillata. A total of 51,304,108 reads were obtained and assembled into 50,536 unigenes. Of these, 21,357 unigenes had homologues in public databases, but the remaining unigenes had no significant matches due to the limited sequence information available and species-specific novel sequences. Functional annotation of the unigenes also revealed general gene expression profile characteristics in the tentacle of C. capillata. A primary goal of this study was to identify putative toxin transcripts. As expected, we screened many transcripts encoding proteins similar to several well-known toxin families including phospholipases, metalloproteases, serine proteases and serine protease inhibitors. In addition, some transcripts also resembled molecules with potential toxic activities, including cnidarian CfTX-like toxins with hemolytic activity, plancitoxin-1, venom toxin-like peptide-6, histamine-releasing factor, neprilysin, dipeptidyl peptidase 4, vascular endothelial growth factor A, angiotensin-converting enzyme-like and endothelin-converting enzyme 1-like proteins. Most of these molecules have not been previously reported in jellyfish. Interestingly, we also characterized a number of transcripts with similarities to proteins relevant to several degenerative diseases, including Huntington's, Alzheimer's and Parkinson's diseases. This is the first description of degenerative disease-associated genes in jellyfish. CONCLUSION: We obtained a well-categorized and annotated transcriptome of C. capillata tentacle that will be an important and valuable resource for further understanding of jellyfish at the molecular level and information on the underlying molecular mechanisms of jellyfish stinging. The findings of this study may also be used in comparative studies of gene expression profiling among different jellyfish species.

Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks.

Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions.

Bioaccessibility versus bioavailability of essential (Cu, Fe, Mn, and Zn) and toxic (Pb) elements from phyto hyperaccumulator Pistia stratiotes: potential risk of dietary intake.

Aquatic weeds are widely used as animal feed in developing countries. However, information about element bioavailability from these plants is lacking. A combination of an in vitro method [physiologically based extraction test (PBET)] and an in vivo feeding trial was used in this study to investigate potential element bioaccessibility and estimated bioavailability of Pistia stratiotes (PS). Cu, Fe, Mn, Zn, and Pb concentrations in PS biomass, artificial gastrointestinal fluids, and rat tissues were determined using atomic absorption spectrometry with electrothermal atomization and inductively coupled plasma-atomic emission spectrometry. PS exhibited elevated Fe, Mn, and Pb levels. The PBET revealed high bioaccessibility of all monitored elements from PS biomass. The results of the in vivo trial were inconsistent with those of the PBET, because animals fed PS exhibited low levels of essential elements in the tissues. The consumption of a PS-supplemented diet significantly decreased total Fe levels and increased the total level of accumulation of Pb in exposed animals. Significantly reduced amounts of essential elements in the intestinal walls indicated a potential disruption in nutrient gastrointestinal absorption in animals fed PS.

Fatty acid solubilizer from the oral disk of the blowfly.

BACKGROUND: Blowflies are economic pests of the wool industry and potential vectors for epidemics. The establishment of a pesticide-free, environmentally friendly blowfly control strategy is necessary. Blowflies must feed on meat in order to initiate the cascade of events that are involved in reproduction including juvenile hormone synthesis, vitellogenesis, and mating. During feeding blowflies regurgitate salivary lipase, which may play a role in releasing fatty acids from triglycerides that are found in food. However, long-chain fatty acids show low solubility in aqueous solutions. In order to solubilize and ingest the released hydrophobic fatty acids, the blowflies must use a solubilizer. METHODOLOGY: We applied native PAGE, Edman degradation, cDNA cloning, and RT-PCR to characterize a protein that accumulated in the oral disk of the black blowfly, Phormia regina. In situ hybridization was carried out to localize the expression at the cellular level. A fluorescence competitive binding assay was used to identify potential ligands of this protein. CONCLUSION: A protein newly identified from P. regina (PregOBP56a) belonged to the classic odorant-binding protein (OBP) family. This gene was expressed in a cluster of cells that was localized between pseudotracheae on the oral disk, which are not accessory cells of the taste peg chemosensory sensilla that normally synthesize OBPs. At pH 7 and pH 6, PregOBP56a bound palmitic, stearic, oleic, and linoleic acids, that are mainly found in chicken meat. The binding affinity of PregOBP56a decreased at pH 5. We propose that PregOBP56a is a protein that solubilizes fatty acids during feeding and subsequently helps to deliver the fatty acids to the midgut where it may help in the process of reproduction. As such, PregOBP56a is a potential molecular target for controlling the blowfly.

The molecular structures of major ampullate silk proteins of the wasp spider, Argiope bruennichi: a second blueprint for synthesizing de novo silk.

The dragline silk of orb-weaving spiders possesses extremely high tensile strength and elasticity. To date, full-length sequences of only two genes encoding major ampullate silk protein (MaSp) in Latrodectus hesperus have been determined. In order to further understand this gene family, we utilized in this study a variety of strategies to isolate full-length MaSp1 and MaSp2 cDNAs in the wasp spider Argiope bruennichi. A. bruennichi MaSp1 and MaSp2 are primarily composed of remarkably homogeneous ensemble repeats containing several complex motifs, and both have highly conserved C-termini and N-termini. Two novel amino acid motifs, GGF and SGR, were found in MaSp1 and MaSp2, respectively. Amino acid composition analysis of silk, luminal contents and predicted sequences indicates that MaSp1 and MaSp2 are two major components of major ampullate glands and that the ratio of MaSp1 to MaSp2 is approximately 3:2 in dragline silk. Furthermore, both the MaSp1:MaSp2 ratio and the conserved termini are closely linked with the production of high quality synthetic fibers. Our results make an important contribution to our understanding of major ampullate silk protein structure and provide a second blueprint for creating new composite silk which mimics natural spider dragline silk.

Cloning, tissue and ontogenetic expression of the taurine transporter in the flatfish Senegalese sole (Solea senegalensis).

Flatfish species seem to require dietary taurine for normal growth and development. Although dietary taurine supplementation has been recommended for flatfish, little is known about the mechanisms of taurine absorption in the digestive tract of flatfish throughout ontogeny. This study described the cloning and ontogenetic expression of the taurine transporter (TauT) in the flatfish Senegalese sole (Solea senegalensis). Results showed a high similarity between TauT in Senegalese sole and other vertebrates, but a change in TauT amino acid sequences indicates that taurine transport may differ between mammals and fish, reptiles or birds. Moreover, results showed that Senegalese sole metamorphosis is an important developmental trigger to promote taurine transport in larvae, especially in muscle tissues, which may be important for larval growth. Results also indicated that the capacity to uptake dietary taurine in the digestive tract is already established in larvae at the onset of metamorphosis. In Senegalese sole juveniles, TauT expression was highest in brain, heart and eye. These are organs where taurine is usually found in high concentrations and is believed to play important biological roles. In the digestive tract of juveniles, TauT was more expressed in stomach and hindgut, indicating that dietary taurine is quickly absorbed when digestion begins and taurine endogenously used for bile salt conjugation may be recycled at the posterior end of the digestive tract. Therefore, these results suggest an enterohepatic recycling pathway for taurine in Senegalese sole, a process that may be important for maintenance of the taurine body levels in flatfish species.

Towards a platform PBPK model to characterize the plasma and tissue disposition of monoclonal antibodies in preclinical species and human.

The objectives of the following investigation were (1) development of a physiologically based pharmacokinetic (PBPK) model capable of characterizing the plasma and tissue pharmacokinetics (PK) of nonspecific or antigen specific monoclonal antibodies (mAbs) in wild type, FcRn knockout, tumor bearing and non tumor bearing mice and (2) evaluation of the scale up potential of the model by characterizing the mouse, rat, monkey and human plasma PK of mAbs, simultaneously. A PBPK model containing 15 tissues, a carcass and a tumor compartment was developed by modifying/augmenting previously published PBPK models. Each tissue compartment was subdivided into plasma, blood cell, endothelial, interstitial and cellular sub-compartments. Each tissue was connected through blood and lymph flow to the systemic circulation. Lymph flow was set to a value 500 times lower than plasma flow and vascular reflection coefficients for each tissue were adjusted according to their vascular pore size. In each tissue endothelial space, mAb entered via pinocytosis and the interaction of FcRn with mAb was described by on and off rates. FcRn bound mAb was recycled and unbound mAb was eliminated by a first order process (K(deg)). The PBPK model was simultaneously fit to the following datasets to estimate four system parameters: (1) plasma and tissue PK of nonspecific mAb in wild type mouse with or without simultaneous intravenous immunoglobulin (IVIG) administration, (2) plasma and tissue PK of nonspecific mAb in FcRn knockout mouse, (3) plasma and tissue PK of nonspecific mAb in tumor bearing mouse, (4) plasma and tissue PK of tumor antigen specific mAb in tumor bearing mouse, and (5) plasma PK of mAb in rat, monkey and human. The model was able to characterize all the datasets reasonably well with a common set of parameters. The estimated value of the four system parameters i.e. FcRn concentration (FcRn), rate of pinocytosis per unit endosomal space (CL(up)), K(deg) and the proportionality constant (C_LNLF) between the rate at which antibody transfers from the lymph node compartment to the blood compartment and the plasma flow of the given species, were found to be 4.98E-05 M (CV% = 11.1), 3.66E-02 l/h/l (%CV = 3.48), 42.9 1/h (%CV = 15.7) and 9.1 (CV% > 50). Thus, a platform PBPK model has been developed that can not only simultaneously characterize mAb disposition data obtained from various previously published mouse PBPK models but is also capable of characterizing mAb disposition in various preclinical species and human.

Purple corn (Zea mays L.) phenolic compounds profile and its assessment as an agent against oxidative stress in isolated mouse organs.

This study was designed to determine the contents of total polyphenols, flavonoids, flavonols, flavanols, and anthocyanins of purple corn (Zea mays L.) extracts obtained with different methanol:water concentrations, acidified with 1% HCl (1 N). Another objective was to determine the antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and deoxyribose assay, individual phenolic compounds by high-performance liquid chromatography (HPLC), and endogenous antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], and total peroxidase [TPX]) activity and lipid peroxidation activity (thiobarbituric acid-reactive substances [TBARS] assay) in isolated mouse organs. Overall, the highest total content of polyphenols, anthocyanins, flavonoids, flavonols, and flavanols was obtained with the 80:20 methanol:water extract, acidified with 1% HCl (1 N). The 50% inhibitory concentration values obtained by the DPPH and ABTS assays with this extract were 66.3 µg/mL and 250 µg/mL, respectively. The antioxidant activity by the FRAP assay was 26.1 µM Trolox equivalents/g, whereas the deoxyribose assay presented 93.6% inhibition. Because of these results, the 80:20 methanol:water extract, acidified with 1% HCl (1 N), was used for the remaining tests. Eight phenolic compounds were identified by HPLC: chlorogenic acid, caffeic acid, rutin, ferulic acid, morin, quercetin, naringenin, and kaempferol. Furthermore, it was observed that the purple corn extract was capable of significantly reducing lipid peroxidation (lower malondialdehyde [MDA] concentrations by the TBARS assay) and at the same time increasing endogenous antioxidant enzyme (CAT, TPX, and SOD) activities in isolated mouse kidney, liver, and brain. On the basis of the results, it was concluded that the purple corn extract contained various bioactive phenolic compounds that exhibited considerable in vitro antioxidant activity, which correlated well with the decreased MDA formation and increase in activity of endogenous antioxidant enzymes observed in the isolated mouse organs. This warrants further in vivo studies with purple corn extracts to assess its antioxidant activity and other bioactivities.

Isolation and characterization of CvIV4: a pain inducing α-scorpion toxin.

BACKGROUND: Among scorpion species, the Buthidae produce the most deadly and painful venoms. However, little is known regarding the venom components that cause pain and their mechanism of action. Using a paw-licking assay (Mus musculus), this study compared the pain-inducing capabilities of venoms from two species of New World scorpion (Centruroides vittatus, C. exilicauda) belonging to the neurotoxin-producing family Buthidae with one species of non-neurotoxin producing scorpion (Vaejovis spinigerus) in the family Vaejovidae. A pain-inducing α-toxin (CvIV4) was isolated from the venom of C. vittatus and tested on five Na(+) channel isoforms. PRINCIPAL FINDINGS: C. vittatus and C. exilicauda venoms produced significantly more paw licking in Mus than V. spinigerus venom. CvIV4 produced paw licking in Mus equivalent to the effects of whole venom. CvIV4 slowed the fast inactivation of Na(v)1.7, a Na(+) channel expressed in peripheral pain-pathway neurons (nociceptors), but did not affect the Na(v)1.8-based sodium currents of these neurons. CvIV4 also slowed the fast inactivation of Na(v)1.2, Na(v)1.3 and Na(v)1.4. The effects of CvIV4 are similar to Old World α-toxins that target Na(v)1.7 (AahII, BmK MI, LqhIII, OD1), however the primary structure of CvIV4 is not similar to these toxins. Mutant Na(v)1.7 channels (D1586A and E1589Q, DIV S3-S4 linker) reduced but did not abolish the effects of CvIV4. CONCLUSIONS: This study: 1) agrees with anecdotal evidence suggesting that buthid venom is significantly more painful than non-neurotoxic venom; 2) demonstrates that New World buthids inflict painful stings via toxins that modulate Na(+) channels expressed in nociceptors; 3) reveals that Old and New World buthids employ similar mechanisms to produce pain. Old and New World α-toxins that target Na(v)1.7 have diverged in sequence, but the activity of these toxins is similar. Pain-inducing toxins may have evolved in a common ancestor. Alternatively, these toxins may be the product of convergent evolution.

Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster.

BACKGROUND: Despite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates. PRINCIPAL FINDINGS: We employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions. CONCLUSIONS: We constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization.

A newly developed DNA microarray is useful to assess induction of cytochromes p450 in the cynomolgus monkey.

Cytochromes P450 (P450s or CYPs) are a gene family of highly homologous genes and include the CYP1-4 family, which is relevant to drug metabolism. In the cynomolgus monkey (which is frequently used in drug metabolism studies), numerous CYPs (mfCYPs) have been identified in the CYP1-4 family. DNA microarrays are useful for high-throughput screening assays; however, there is a potential problem with cross-hybridization of highly homologous genes in the gene family. This problem might be solved with the use of low-density DNA microarrays, with which specific validation can be performed for the genes on the microarray. We have developed a DNA microarray for the 20 mfCYPs and have evaluated and validated its specificity and usefulness. First, in both DNA microarray and quantitative polymerase chain reaction (qPCR) analyses, hepatic expression of each mfCYP correlated well, and similar tissue expression patterns were observed for five representative mfCYPs, confirming the specificity of the DNA microarray. Second, the usefulness of this DNA microarray was validated by induction analysis of mfCYPs in primary hepatocytes, which successfully detected known responders, but also novel responders (mfCYP2C43, mfCYP2C75, and mfCYP3A5 for rifampicin), as confirmed by qPCR analysis. This DNA microarray can thus be utilized for high-throughput assays during drug development.

Short chain saturated fatty acids decrease circulating cholesterol and increase tissue PUFA content in the rat.

This study investigates the effect of various dietary saturated fatty acid (SFA) profiles on plasma lipid parameters and tissue fatty acid composition in rats. The experiment was designed to monitor polyunsaturated fatty acids (PUFA) levels, while examining different amounts and types of SFA. Four isocaloric diets were prepared, containing 10-11 mol% of fatty acids (FA) as linoleic acid (LNA) and 2.5 mol% as α-linolenic acid (ALA), leading to an identical and well-balanced LNA/ALA ratio. The initial rapeseed oil/corn oil mixture providing ALA and LNA was enriched with olive oil to prepare the olive oil diet. The butterfat diet was supplemented with butterfat, containing short-chain SFA (C4:0-C10:0, 17 mol% of FA), lauric acid (C12:0, 3.2 mol%), myristic acid (C14:0, 10.5 mol%) and palmitic acid (C16:0, 14.5 mol%). The saturates diet was supplemented with trilaurin, trimyristin and tripalmitin to obtain the same level of lauric, myristic and palmitic acids as the butterfat diet, without the short-chain SFA. The trimyristin diet was enriched with trimyristin only. The results showed that the butterfat diet contributed to specific effects, compared to the olive oil diet and the saturates and trimyristin diets: a decrease in plasma total, LDL- and HDL-cholesterol, higher tissue storage of ALA and LNA, and a higher level of (n-3) highly unsaturated fatty acids in some tissues. This study supports the hypothesis that in diets with identical well-balanced LNA/ALA ratios, short chain SFA may decrease circulating cholesterol and increase tissue polyunsaturated fatty acid content in the rat.

An extract of the pleurotus ostreatus mushroom bolsters the glutathione redox system in various organs of aged rats.

This study is to investigate the putative effect of an extract of the oyster mushroom, Pleurotus ostreatus, on reduced glutathione (GSH) and its metabolic enzymes in major organs of male albino rats. A significant (P < .05) decrease in the level of GSH was observed in liver, kidneys, heart, and brain of aged rats compared to young rats. Activities of glutathione S-transferase (GST), glutathione reductase (GR), and glucose 6-phosphate dehydrogenase (G6PDH) were significantly (P < .05) lower in the liver, kidneys, heart, and brain of aged rats. The isoform pattern of these enzymes in aged rats also revealed variations in relative concentrations, presumably due to oxidative stress during aging. Administration of the extract of P. ostreatus to aged rats resulted in a significant (P < .05) increase in the levels of GSH and elevated activities of GST, GR, and G6PDH in liver, kidney, heart, and brain tissues. An increased staining intensity of isoforms of GST and G6PDH was also noted in aged rats that had been treated with the mushroom extract compared to aged untreated rats. The results of this study may suggest that an extract of P. ostreatus, a potential antioxidant, can prevent the oxidation of GSH and protect its related enzymes during aging.