Genomics, evolution and biological functions of the pacifastin peptide family: a conserved serine protease inhibitor family in arthropods

The last decade, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5). The light chain of the heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of six conserved cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-6)-Cys-Thr-Xaa(3)-Cys) with the locust inhibitors. Via cDNA cloning, eight pacifastin-related precursors have been identified in locusts. Interestingly, additional pacifastin-related precursors have been identified in Diptera, Lepidoptera and Coleoptera utilising an in silico data mining approach.     

DNA-Dependent protein kinase is not required for efficient lentivirus integration

How DNA is repaired after retrovirus integration is not well understood. DNA-dependent protein kinase (DNA-PK) is known to play a central role in the repair of double-stranded DNA breaks. Recently, a role for DNA-PK in retroviral DNA integration has been proposed (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Reduced transduction efficiency and increased cell death by apoptosis were observed upon retrovirus infection of cultured scid cells. We have used a human immunodeficiency virus (HIV) type 1 (HIV-1)-derived lentivirus vector system to further investigate the role of DNA-PK during integration. We measured lentivirus transduction of scid mouse embryonic fibroblasts (MEF) and xrs-5 or xrs-6 cells. These cells are deficient in the catalytic subunit of DNA-PK and in Ku, the DNA-binding subunit of DNA-PK, respectively. At low vector titers, efficient and stable lentivirus transduction was obtained, excluding an essential role for DNA-PK in lentivirus integration. Likewise, the efficiency of transduction of HIV-derived vectors in scid mouse brain was as efficient as that in control mice, without evidence of apoptosis. We observed increased cell death in scid MEF and xrs-5 or xrs-6 cells, but only after transduction with high vector titers (multiplicity of infection [MOI], >1 transducing unit [TU]/cell) and subsequent passage of the transduced cells. At an MOI of <1 TU/cell, however, transduction efficiency was even higher in DNA-PK-deficient cells than in control cells. Taken together, the data suggest a protective role of DNA-PK against cellular toxicity induced by high levels of retrovirus integrase or integration. Another candidate cellular enzyme that has been claimed to play an important role during retrovirus integration is poly(ADP-ribose) polymerase (PARP). However, no inhibition of lentivirus vector-mediated transduction or HIV-1 replication by 3-methoxybenzamide, a known PARP inhibitor, was observed. In conclusion, DNA-PK and PARP are not essential for lentivirus integration.     

Development of a real-time PCR assay for measurement of yellow protein mRNA transcription in the desert locust Schistocerca gregaria: a basis for isolation of a peptidergic regulatory factor

A major unresolved issue in insect endocrinology concerns the question of whether or not insects have sex hormones. Conclusive evidence in favor of the presence of such hormones awaits the establishment of appropriate bioassays in males. The cuticle of sexually mature males of the desert locust Schistocerca gregaria turns yellow in gregarious conditions only. Neither females nor isolated males ever turn yellow. The yellowing is due to the deposition in the cuticle of a male-specific Yellow Protein (YP), of which the amino acid sequence is known. In this paper, we describe the partial cloning of the cDNA encoding this Yellow Protein. The tissue distribution and temporal expression of the YP-mRNA is studied in detail using RT-PCR. Furthermore, an RT-PCR based bioassay was developed, which may serve as a reliable tool to help identify the hormones controlling the yellowing process. In addition to juvenile hormone, we have shown that a factor present in the brain-corpora cardiaca is involved in the yellow coloration, as injection of an extract induces the expression of YP-mRNA in isolated gregarious males.     

Hsmar1 Transposition Is Sensitive to the Topology of the Transposon Donor and the Target

Hsmar1 is a member of the Tc1-mariner superfamily of DNA transposons. These elements mobilize within the genome of their host by a cut-and-paste mechanism. We have exploited the in vitro reaction provided by Hsmar1 to investigate the effect of DNA supercoiling on transposon integration. We found that the topology of both the transposon and the target affect integration. Relaxed transposons have an integration defect that can be partially restored in the presence of elevated levels of negatively supercoiled target DNA. Negatively supercoiled DNA is a better target than nicked or positively supercoiled DNA, suggesting that underwinding of the DNA helix promotes target interactions. Like other Tc1-mariner elements, Hsmar1 integrates into 5′-TA dinucleotides. The direct vicinity of the target TA provides little sequence specificity for target interactions. However, transposition within a plasmid substrate was not random and some TA dinucleotides were targeted preferentially. The distribution of intramolecular target sites was not affected by DNA topology.     

Molecular phylogeny of Malenchus and Filenchus (Nematoda: Tylenchidae)

The family Tylenchidae is phylogenetically important to understanding early‐branching Tylenchomorpha and to assess soil ecosystems. In this study, we focus on Malenchus and Filenchus as representatives of the Tylenchidae. Samples collected worldwide result in 58 new sequences, and light microscopy and transmission electron microscopy provide details on morphological features. For the first time, comprehensive morphological data are evaluated in the context of a molecular framework, thus highlighting the phylogenetic and evolutionary complexity of this structurally minimalistic group. Results show that the genus Filenchus is polyphyletic in both the 18S and 28S rDNA phylogeny, while Malenchus is polyphyletic and monophyletic in the 28S rDNA and the 18S rDNA, respectively. Ultrastructural study demonstrates specific aspects of lateral cuticular incisures, cuticular layering and the amphideal fovea are surprisingly congruent with the obtained molecular phylogenies, while classical characteristics such as cuticle annulations are evolutionary highly plastic and mosaic in distribution. The study also reveals the shortage of D2/D3 domain in 28S rDNA as a phylogenetic marker for early‐branching Tylenchomorpha.     

Structure,development, and evolutive patterns of spermatozoa in rhabditid nematodes (Nematoda: Rhabditida)

Spermatogenesis of five rhabditid nematodes was studied using transmission electron microscopy and is described herein. Structure and development of nematode sperm in all available representatives of the extensive order Rhabditida have been analysed and the main characteristics of each infraorder are discussed. The ancestral sperm of the order Rhabditida was reconstructed using maximum likelihood and Bayesian methods based on 44 ultrastructural sperm characters. The hypothetical ancestral spermatogenesis of the order Rhabditida agrees with the previously suggested “rhabditid” pattern and appears to be conserved throughout the order Rhabditida. Despite the enormous variation of rhabditid nematodes, few groups deviate from the ancestral pattern. This conserved pattern can be informative within the phylum Nematoda at order level, but poses limitations when used in taxonomic and phylogenetic analysis within Rhabditida.