Moult cycle specific differential gene expression profiling of the crab Portunus pelagicus

Background: Crustacean moulting is a complex process involving many regulatory pathways. A holistic approach to examine differential gene expression profiles of transcripts relevant to the moulting process, across all moult cycle stages, was used in this study. Custom cDNA microarrays were constructed for Portunus pelagicus. The printed arrays contained 5000 transcripts derived from both the whole organism, and from individual organs such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages.Results: A total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 175 singletons and 62 contigs, resulting in 237 unique putative genes. The gene sequences were classified into the following biological functions: cuticular proteins associated with arthropod exoskeletons, farnesoic acid O-methyltransferase (FaMeT), proteins belonging to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators and ribosomal proteins. Moult cycle-related differential expression patterns were observed for many transcripts. Of particular interest were those relating to the formation and hardening of the exoskeleton, and genes associated with cell respiration and energy metabolism.Conclusions: The expression data presented here provide a chronological depiction of the molecular events associated with the biological changes that occur during the crustacean moult cycle. Tracing the temporal expression patterns of a large variety of transcripts involved in the moult cycle of P. pelagicus can provide a greater understanding of gene function, interaction, and regulation of both known and new genes with respect to the moulting process

Unrecorded alcohol consumption in Russia: toxic denaturants and disinfectants pose additional risks

In 2005, 30% of all alcohol consumption in Russia was unrecorded. This paper describes the chemical composition of unrecorded and low cost alcohol, including a toxicological evaluation. Alcohol products (n=22) from both recorded and unrecorded sources were obtained from three Russian cities (Saratov, Lipetsk and Irkutsk) and were chemically analyzed. Unrecorded alcohols included homemade samogons, medicinal alcohols and surrogate alcohols. Analysis included alcoholic strength, levels of volatile compounds (methanol, acetaldehyde, higher alcohols), ethyl carbamate, diethyl phthalate (DEP) and polyhexamethyleneguanidine hydrochloride (PHMG). Single samples showed contamination with DEP (275–1269 mg/l) and PHMG (515 mg/l) above levels of toxicological concern. Our detailed chemical analysis of Russian alcohols showed that the composition of vodka, samogon and medicinal alcohols generally did not raise major public health concerns other than for ethanol. It was shown, however, that concentration levels of DEP and PHMG in some surrogate alcohols make these samples unfit for human consumption as even moderate drinking would exceed acceptable daily intakes

Clinical isolates of the modern Mycobacterium tuberculosis lineage 4 evade host defense in human macrophages through eluding IL-1\u3b2-induced autophagy article

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected over 1.7 billion people worldwide and causes 1.4 million deaths annually. Recently, genome sequence analysis has allowed the reconstruction of Mycobacterium tuberculosis complex (MTBC) evolution, with the identification of seven phylogeographic lineages: four referred to as evolutionarily "ancient", and three "modern". The MTBC strains belonging to "modern" lineages appear to show enhanced virulence that may have warranted improved transmission in humans over ancient lineages through molecular mechanisms that remain to be fully characterized. To evaluate the impact of MTBC genetic diversity on the innate immune response, we analyzed intracellular bacterial replication, inflammatory cytokine levels, and autophagy response in human primary macrophages infected with MTBC clinical isolates belonging to the ancient lineages 1 and 5, and the modern lineage 4. We show that, when compared to ancient lineage 1 and 5, MTBC strains belonging to modern lineage 4 show a higher rate of replication, associated to a significant production of proinflammatory cytokines (IL-1\u3b2, IL-6, and TNF-\u3b1) and induction of a functional autophagy process. Interestingly, we found that the increased autophagic flux observed in macrophages infected with modern MTBC is due to an autocrine activity of the proinflammatory cytokine IL-1\u3b2, since autophagosome maturation is blocked by an interleukin-1 receptor antagonist. Unexpectedly, IL-1\u3b2-induced autophagy is not disadvantageous for the survival of modern Mtb strains, which reside within Rab5-positive phagosomal vesicles and avoid autophagosome engulfment. Altogether, these results suggest that autophagy triggered by inflammatory cytokines is compatible with a high rate of intracellular bacilli replication and may therefore contribute to the increased pathogenicity of the modern MTBC lineages

Beta-HPV 5 and 8 E6 Promote p300 Degradation by Blocking AKT/p300 Association

The E6 oncoprotein from high-risk genus alpha human papillomaviruses (α-HPVs), such as HPV 16, has been well characterized with respect to the host-cell proteins it interacts with and corresponding signaling pathways that are disrupted due to these interactions. Less is known regarding the interacting partners of E6 from the genus beta papillomaviruses (β-HPVs); however, it is generally thought that β-HPV E6 proteins do not interact with many of the proteins known to bind to α-HPV E6. Here we identify p300 as a protein that interacts directly with E6 from both α- and β-HPV types. Importantly, this association appears much stronger with β-HPV types 5 and 8-E6 than with α-HPV type 16-E6 or β-HPV type 38-E6. We demonstrate that the enhanced association between 5/8-E6 and p300 leads to p300 degradation in a proteasomal-dependent but E6AP-independent manner. Rather, 5/8-E6 inhibit the association of AKT with p300, an event necessary to ensure p300 stability within the cell. Finally, we demonstrate that the decreased p300 protein levels concomitantly affect downstream signaling events, such as the expression of differentiation markers K1, K10 and Involucrin. Together, these results demonstrate a unique way in which β-HPV E6 proteins are able to affect host-cell signaling in a manner distinct from that of the α-HPVs

Isolation and characterization of the complete cDNA sequence encoding a putative insulin-like peptide from the androgenic gland of Penaeus monodon

Sexual differentiation in male crustaceans is known to be controlled by the androgenic gland (AG), possibly through a peptide hormone. Recently, three freshwater and one marine crustacean decapod genes encoding AG-specific insulin-like peptides were characterized. We report here the molecular cloning of the complete sequence encoding an AG-specific insulin-like peptide (Pm-IAG) in the commercially important marine Penaeid prawn, Penaeus monodon. The deduced precursor sequence consists of a signal peptide, B chain, C peptide and an A chain. It exhibits the same structural organization as that of previously identified crustacean insulin-like androgenic gland specific peptides (IAGs). The positions of cysteine residues of the putative A and B chains, which govern the folding of the mature peptide via the formation of disulfide bridges, are highly conserved among the prawn and other crustaceans, while the rest of the amino acids show low sequence similarity. Gene expression analysis of Pm-IAG in several tissues, including the closely juxtaposed sperm duct and muscle, confirmed that it is specifically expressed in the AG. The findings suggest that with an appropriate intervention, sexual differentiation could be manipulated and thus might be instrumental for the establishment of monosex culture in this bimodally growing shrimp

Isolation and expression analysis of multiple isoforms of putative farnesoic acid O-methyltransferase in several crustacean species

Farnesoic acid O-methyltransferase (FaMeT) is the enzyme responsible for the conversion of farnesoic acid (FA) to methyl farnesoate (ME) in the final step of ME synthesis. Multiple isoforms of putative FaMeT were isolated from six crustacean species belonging to the families Portunidae, Penacidae, Scyllaridae and Parastacidae. The portunid crabs Portunus pelagicus and Scylla serrata code for three forms: short, intermediate and long. Two isoforms (short and long) were isolated from the penaeid prawns Penaeus monodon and Fenne-ropenaeus merguiensis. Two isoforms were also identified in the scyllarid Thenus orientalis and parastacid Cherax quadricarinatus. Putative FaMeT sequences were also amplified from the genomic DNA of P. pelagicus and compared to the putative FaMeT transcripts expressed. Each putative FaMeT cDNA isoform was represented in the genomic DNA, indicative of a multi-gene family. Various tissues from P. pelagicus were individually screened for putative FaMeT expression using PCR and fragment analysis. Each tissue type expressed all three isoforms Of putative FaMeT irrespective of sex or moult stage. Protein domain analysis revealed the presence of a deduced casein kinase II phosphorylation site present only in the long isoform of putative FaMeT. Crown copyright (c) 2006 Published by Elsevier Inc. All rights reserved

Molecular Characterisation of Colour Formation in the Prawn <em>Fenneropenaeus merguiensis</em>

<div><p>Introduction</p><p>Body colouration in animals can have a range of functions, with predator protection an important aspect of colour in crustaceans. Colour determination is associated with the carotenoid astaxanthin, which is taken up through the diet and stabilised in the tissues by the protein crustacyanin. As a variety of genes are found to play a role in colour formation in other systems, a holistic approach was employed in this study to determine the factors involved in <i>Fenneropenaeus merguiensis</i> colouration.</p> <p>Results</p><p>Full length <i>F. merguiensis</i> crustacyanin subunit A and C sequences were isolated. Crustacyanin subunit A and C were found in the <i>F. merguiensis</i> transcriptomes of the muscle/cuticle tissue, hepatopancreas, eye stalk and nervous system, using 454 next generation sequencing technology. Custom microarray analysis of albino, light and dark <i>F. merguiensis</i> cuticle tissue showed genes encoding actin, sarcoplasmic calcium-binding protein and arginine kinase to be 4-fold or greater differentially expressed (<i>p</i><0.05) and down-regulated in albinos when compared to light and dark samples. QPCR expression analysis of crustacyanin and total astaxanthin pigment extraction revealed significantly (<i>p</i><0.05) lower crustacyanin subunit A and C gene transcript copy numbers and total astaxanthin levels in albinos than in the light and dark samples. Additionally, crustacyanin subunit A and C expression levels correlated positively with each other.</p> <p>Conclusions</p><p>This study identified gene products putatively involved in crustacean colouration, such as crustacyanin, sarcoplasmic calcium-binding protein and forms of actin, and investigated differences in gene expression and astaxanthin levels between albino, light and dark coloured prawns. These genes open a path to enhance our understanding of the biology and regulation of colour formation.</p> </div