Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile

Background

Schistosoma mansoni is the major causative agent of schistosomiasis. The parasite takes advantage of host signals to complete its development in the human body. Tumor necrosis factor-alpha (TNF-α) is a human cytokine involved in skin inflammatory responses, and although its effect on the adult parasite''s metabolism and egg-laying process has been previously described, a comprehensive assessment of the TNF-α pathway and its downstream molecular effects is lacking.

Methodology/Principal Findings

In the present work we describe a possible TNF-α receptor (TNFR) homolog gene in S. mansoni (SmTNFR). SmTNFR encodes a complete receptor sequence composed of 599 amino acids, and contains four cysteine-rich domains as described for TNFR members. Real-time RT-PCR experiments revealed that SmTNFR highest expression level is in cercariae, 3.5 (±0.7) times higher than in adult worms. Downstream members of the known human TNF-α pathway were identified by an in silico analysis, revealing a possible TNF-α signaling pathway in the parasite. In order to simulate parasite''s exposure to human cytokine during penetration of the skin, schistosomula were exposed to human TNF-α just 3 h after cercariae-to-schistosomula in vitro transformation, and large-scale gene expression measurements were performed with microarrays. A total of 548 genes with significantly altered expression were detected, when compared to control parasites. In addition, treatment of adult worms with TNF-α caused a significantly altered expression of 1857 genes. Interestingly, the set of genes altered in adults is different from that of schistosomula, with 58 genes in common, representing 3% of altered genes in adults and 11% in 3 h-old early schistosomula.

Conclusions/Significance

We describe the possible molecular elements and targets involved in human TNF-α effect on S. mansoni, highlighting the mechanism by which recently transformed schistosomula may sense and respond to this host mediator at the site of cercarial penetration into the skin.     

Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis

In bivalve molluscs including oysters, lysozymes play an important role in the host defense mechanisms against invading microbes. However, it remains unclear in which sites/cells the lysozyme genes are expressed and which subsequently produced the enzyme. This study cloned lysozyme cDNAs from the digestive organs of Pacific oyster Crassostrea gigas and European flat oyster Ostrea edulis. Both complete sequences of two oysters' lysozymes were composed of 137 amino acids. Two translated proteins present a high content in cysteine residues. Phylogenetic analyses showed that these oysters' lysozymes clustered with the invertebrate-type lysozymes of other bivalve species. In the Pacific oyster, lysozyme mRNA was expressed in all tissues except for those of the adductor muscle. In situ hybridization analyses revealed that lysozyme mRNA was expressed strongly in basophil cells in the digestive gland tubule of C. gigas, but not in digestive cells. Results indicated that the basophil cells of the oyster digestive gland are the sites of lysozyme synthesis.     

Cloning of a Novel Rat Gene, DB83, That Encodes a Putative Membrane Protein

Using a partial cDNA sequence and a 5'-RACE technique, we isolateda novel cDNA from rat liver referred to as DB83. DB83 had fourhydrophobic trans-membrane domains and one N-myristoylationsite as well as multiple possible phosphorylation sites. Thedb83 gene was highly expressed in the liver and significantlyin brain, lungs and kidneys. We suggest that DB83 is a tissue-specificputative membrane protein.     

Determination of absolute configuration of 1,3-diols by the modified Mosher's method using their di-MTPA esters.

1,3-Diols are frequently involved in biologically important compounds and, therefore, determination of the stereochemistry of these structural elements, in particular those in acyclic systems, has been one of the focuses of attention in natural products chemistry. The modified Mosher's method, commonly used for the determination of the absolute configuration of secondary alcohols, was applied to determine the absolute configuration of 1,3-diols with their di-MTPA esters. Several epimeric pairs of syn- and anti-1,3-diols with known absolute configurations were converted to the corresponding di-MTPA esters and the iDelta;delta values were then calculated. For the acyclic syn-1,3-diols, the iDelta;delta values were systematically arranged as predicted from the basic concept of the modified Mosher's method, demonstrating that the method is valid for these compounds. In contrast, the iDelta;delta values were irregularly arranged for the acyclic anti-1,3-diols and, accordingly, this method is not valid for these cases. These results are complementary to those of the previously reported CD exciton chirality method and, hence, the combined use of the modified Mosher's method and the CD exciton chirality method can determine the absolute configuration of the acyclic 1,3-diols. Also, this method is successfully applicable to cyclic 1,3-diols irrespective of their relative stereochemistry.     

2,2-Functionalized analogues of 1alpha,25-dihydroxyvitamin D3, the potent inducers of cell differentiation

All four possible A-ring stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin D(3) (4) were designed and convergently synthesized. Nine-step conversion of methyl hydroxypivalate 6 provided the desired A-ring enyne synthon (13a,b) in good overall yield. Cross-coupling reaction of the A-ring synthon 13a,b with the CD-ring portion in the presence of palladium catalyst, followed by deprotection, gave the vitamin analogues (4a-d). We also synthesized four stereoisomers of 2,2-ethano-1,25-dihydroxyvitamin D(3) (5), as novel spiro-ring analogues having cyclopropane fused at the C2 position. Biological potencies of the synthesized compounds were assessed in terms of the vitamin D receptor (VDR) binding affinity, as well as the HL-60 cell differentiation-inducing activity. The 2,2-ethano analogue 5a showed a comparable activity to the natural hormone 1, while the 2,2-dimethyl analogue 4a exhibited one-third of the activity of 1 in cell differentiation, with the reduced VDR binding affinity.     

Phylogenetic analysis of archaeal PCNA homologues

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27 kDa) and 248 aa (27 kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages. Received: July 10, 2000 / Accepted: September 26, 2000     

Light effects on cell development and secondary metabolism in Monascus

In nature, light is one of most crucial environmental signals for developmental and physiological processes in various organisms, including filamentous fungi. We have found that both red light and blue light affect development in Monascus, influencing the processes of mycelium and spore formation, and the production of secondary metabolites such as -aminobutyric acid, red pigments, monacolin K and citrinin. Additionally, we observed that the wavelength of light affects these developmental and physiological processes in different ways. These findings suggest that Monascus possesses a system for differential light response and regulation.