Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2)

During influenza virus infection, viral ribonucleoproteins (vRNPs) are replicated in the nucleus and must be exported to the cytoplasm before assembling into mature viral particles. Nuclear export is mediated by the cellular protein Crm1 and putatively by the viral protein NEP/NS2. Proteolytic cleavage of NEP defines an N-terminal domain which mediates RanGTP-dependent binding to Crm1 and a C-terminal domain which binds to the viral matrix protein M1. The 2.6 A crystal structure of the C-terminal domain reveals an amphipathic helical hairpin which dimerizes as a four-helix bundle. The NEP-M1 interaction involves two critical epitopes: an exposed tryptophan (Trp78) surrounded by a cluster of glutamate residues on NEP, and the basic nuclear localization signal (NLS) of M1. Implications for vRNP export are discussed.     

A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium

Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSBDelta. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBDelta histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanSB, VanSBDelta and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBDelta histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBDelta was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d-alanyl-d-alanine ending pentapeptide and to constitutive synthesis of d-alanyl-d-lactate terminating peptidoglycan precursors, following loss of d-alanine:d-alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a 'slippage' type of genetic rearrangement in VanB-type strains.     

Interstitial cells of Cajal in the striated musculature of the mouse esophagus

A key feature of signal processing in the mammalian retina is parallel processing, where the segregation of visual information, e.g., brightness, darkness, and color, starts at the first synapse in the retina, the photoreceptor synapse. These various aspects are transmitted in parallel from the input neurons of the retina, the photoreceptor cells, through the interconnecting bipolar cells, to the output neurons, the ganglion cells. The photoreceptors and bipolar cells release a single excitatory neurotransmitter, glutamate, at their synapses. This parsimony is contrasted by the expression of a plethora of glutamate receptors, receptor subunits, and isoforms. The detailed knowledge of the synaptic distribution of glutamate receptors thus is of major importance in understanding the mechanisms of retinal signal processing. This review intends to highlight recent studies on the distribution of glutamate receptors at the photoreceptor synapses of the mammalian retina.     

In Vivo Evidence That the Stromelysin-3 Metalloproteinase Contributes in a Paracrine Manner to Epithelial Cell Malignancy

Stromelysin-3 (ST3; Basset, P., J.P. Bellocq, C. Wolf, I. Stoll, P. Hutin, J.M. Limacher, O.L. Podhajcer, M.P. Chenard, M.C. Rio, P. Chambon. 1990. Nature. 348:699–704) is a matrix metalloproteinase (MMP) expressed in mesenchymal cells located close to epithelial cells, during physiological and pathological tissue remodeling processes. In human carcinomas, high ST3 levels are associated with a poor clinical outcome, suggesting that ST3 plays a role during malignant processes. In this study we report the ST3 gene inactivation by homologous recombination. Although ST3 null mice (ST3^−/−) were fertile and did not exhibit obvious alterations in appearance and behavior, the lack of ST3 altered malignant processes. Thus, the suppression of ST3 results in a decreased 7,12-dimethylbenzanthracene-induced tumorigenesis in ST3^−/− mice. Moreover, ST3^−/− fibroblasts have lost the capacity to promote implantation of MCF7 human malignant epithelial cells in nude mice (P < 0.008). Finally, we show that this ST3 paracrine function requires extracellular matrix (ECM)-associated growth factors. Altogether, these findings give evidence that ST3 promotes, in a paracrine manner, homing of malignant epithelial cells, a key process for both primary tumors and metastases. Therefore, ST3 represents an appropriate target for specific MMP inhibitor(s) in future therapeutical approaches directed against the stromal compartment of human carcinomas.     

Dedicated Roles of Plastid Transketolases during the Early Onset of Isoprenoid Biogenesis in Pepper Fruits

Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B₁), and pyridoxine (vitamin B₆) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.