Assignment of 1H, 13C, and 15N resonances of YgiT, a putative DNA interacting protein from E. coli, containing one HTH and two CxxC motifs

The 131 residues protein encoded by the open reading frame ygiT of E. coli contains two characteristic domains: a zinc finger protein-like structure with two CxxC motives at its N-terminus and a helix-turn-helix (HTH) motif at its C-terminus. We report the backbone and side chain ¹H, ¹³C, and ¹⁵N resonances assignment of YgiT.     

Stable expression of a retrovirally transferred adhesion molecule in a human melanoma-specific cytotoxic T lymphocyte clone

 The adoptive transfer of in vitro generated tumor-specific cytotoxic T lymphocytes (CTL) is considered a promising perspective in cancer therapy. One possible drawback lies in the inappropriate homing of in vitro cultured lymphocytes, which could be circumvented by introducing the appropriate targeting molecules. Here we describe a protocol that allows a rapid and stable transfection of cytotoxic T cell clones. As a model system we used a CTL clone specific for the melanoma-associated antigen gp100 and a cDNA encoding for murine CD14 containing the variant exen v10 which is supposed to facilitate lymphocyte homing towards the skin. CD44v10 cDNA was ligated into the retroviral vector pMV-7, which was used to transfect the ecotropic GP-E-86 and the amphotropic PA317 cells. After several cycles of transduction to increase the viral titre, supernatants of the amphotropic PA317-CD44v10 line were used for transduction of CD44v10 into a human CTL clone. After three cycles of transduction at 12-h intervals, low but stable expression of CD44v10 was observed throughout the culture period of 10 weeks. The phenotype of the transduced CTL clone was unaltered and the cytotoxic potential was only slightly reduced as compared to the parental clone. The efficiency of stable transduction within a period of 1 week makes the protocol well suited for the in vivo transfer of transduced cells and, in the special case, should guarantee appropriate homing of the transduced CTL clone. Received: 14 August 1997 / Accepted: 20 November 1997     

A molecular‐genetic understanding of diapause in spider mites: current knowledge and future directions

During unfavourable conditions, many arthropods have the ability to enter into diapause and synchronize their development and reproduction to seasonal patterns. Diapause or winter hibernation in insects and mites is set off by a number of cues, with photoperiod being the most well‐defined and strongest signal. This review focuses on the current knowledge of ‘‐omics’ data and the genetics of diapause in the two‐spotted spider mite Tetranychus urticae, a member of the family Tetranychidae (Arthropoda: Chelicerata: Arachnida: Acari). This species is a serious polyphagous pest and females undergo a reproductive facultative diapause when immature stages are exposed to long nights. Winter hibernation induces different physiological processes characterized by a metabolic suppression, different energy use, increased stress tolerance and the production of cryoprotectants, all initiated by a complex signal transduction pathway. Keto‐carotenoids are known to cause the deeply orange colour typical for diapausing females. Furthermore, research with colour mutants of T. urticae has shown the need for carotenoids with respect to the induction of diapause, even though the molecular‐genetic mechanisms underlying these colour phenotypes are still unknown. In addition, marked latitudinal variation in diapause incidence among populations has been observed in nature, with modes of inheritance ranging from recessive to dominant, as well as monogenic to polygenic. We end by highlighting the emerging opportunities for functional studies that aim to unravel the complex factors underlying diapause in spider mites.     

Ribonucleotide reductase inhibition by p-alkoxyphenols studied by molecular docking and molecular dynamics simulations

Ribonucleotide reductase (RNR) is necessary for production of the precursor deoxyribonucleotides for DNA synthesis. Class Ia RNR functions via a stable free radical in one of the two components protein R2. The enzyme mechanism involves long range (proton coupled) electron transfer between protein R1 and the tyrosyl radical in protein R2. Earlier experimental studies showed that p-alkoxyphenols inhibit RNR. Here, molecular docking and molecular dynamics simulations involving protein R2 suggest an inhibition mechanism for p-alkoxyphenols . A low energy binding pocket is identified in protein R2. The preferred configuration provides a structural basis explaining their specific binding to the Escherichia coli and mouse R2 proteins. Trp48 (E. coli numbering), on the electron transfer pathway, is involved in the interactions with the inhibitors. The relative order of the binding energies calculated for the phenol derivatives to protein R2 is correlated with earlier experimental data on inhibition efficiency, in turn related to increasing size of the hydrophobic alkyl substituents. Using the configuration identified by molecular docking as a starting point for molecular dynamics simulations, we find that the p-allyloxyphenol interrupts the catalytic electron transfer pathway of the R2 protein by forming hydrogen bonds with Trp48 and Asp237, thus explaining the inhibitory activity of p-alkoxyphenols.     

Organization of the pronephric kidney revealed by large-scale gene expression mapping

Background

The pronephros, the simplest form of a vertebrate excretory organ, has recently become an important model of vertebrate kidney organogenesis. Here, we elucidated the nephron organization of the Xenopus pronephros and determined the similarities in segmentation with the metanephros, the adult kidney of mammals.

Results

We performed large-scale gene expression mapping of terminal differentiation markers to identify gene expression patterns that define distinct domains of the pronephric kidney. We analyzed the expression of over 240 genes, which included members of the solute carrier, claudin, and aquaporin gene families, as well as selected ion channels. The obtained expression patterns were deposited in the searchable European Renal Genome Project Xenopus Gene Expression Database. We found that 112 genes exhibited highly regionalized expression patterns that were adequate to define the segmental organization of the pronephric nephron. Eight functionally distinct domains were discovered that shared significant analogies in gene expression with the mammalian metanephric nephron. We therefore propose a new nomenclature, which is in line with the mammalian one. The Xenopus pronephric nephron is composed of four basic domains: proximal tubule, intermediate tubule, distal tubule, and connecting tubule. Each tubule may be further subdivided into distinct segments. Finally, we also provide compelling evidence that the expression of key genes underlying inherited renal diseases in humans has been evolutionarily conserved down to the level of the pronephric kidney.

Conclusion

The present study validates the Xenopus pronephros as a genuine model that may be used to elucidate the molecular basis of nephron segmentation and human renal disease.