Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by the gene that is defective in cystic fibrosis, the most common lethal inherited disease among the Caucasian population. CFTR belongs to the ABC transporter superfamily, whose members form macromolecular architectures composed of two membrane-spanning domains and two nucleotide-binding domains (NBDs). The experimental structures of NBDs from several ABC transporters have recently been solved, opening new avenues for understanding the structure/function relationships and the consequences of some disease-causing mutations of CFTR. Based on a detailed sequence/structure analysis, we propose here a three-dimensional model of the human CFTR NBD heterodimer. This model, which is in agreement with recent experimental data, highlights the specific features of the CFTR asymmetric active sites located at the interface between the two NBDs. Moreover, additional CFTR-specific features can be identified at the subunit interface, which may play critical roles in active site interdependence and are uncommon in other NBD dimers.Received 16 October 2003; received after revision 16 November 2003; accepted 21 November 2003     

Molecular aspects of pathogenesis in osteoarthritis: the role of inflammation

Arthritic diseases cause enormous burdens in terms of pain, crippling, and disability. Osteoarthritis (OA), the most common form of arthritis, is characterized by a slow progressive degeneration of articular cartilage. The exact etiology of OA is not known, but the degradation of cartilage matrix components is generally agreed to be due to an increased synthesis and activation of extracellular proteinases, mainly matrix metalloproteinases. Insufficient synthesis of new matrix macromolecules is also thought to be involved, possibly as a consequence of deficient stimulation by growth factors. Although OA is defined as a noninflammatory arthropathy, proinflammatory cytokines such as interleukin-1 have been implicated as important mediators in the disease. In response to interleukin-1, chondrocytes upregulate the production of nitric oxide and prostaglandin E₂, two factors that have been shown to induce a number of the cellular changes associated with OA. The generation of these key signal molecules depends on inducible enzymes and can be suppressed by pharmacological inhibitors.     

Cellular and molecular aspects of drugs of the future: oxaliplatin

Oxaliplatin (Eloxatine) is a third-generation platinum compound which has shown a wide antitumour effect both in vitro and in vivo, a better safety profile than cisplatin and a lack of cross-resistance with cisplatin and carboplatin. In this scenario, oxaliplatin may represent an innovative and challenging drug extending the antitumour activity in diseases such as gastrointestinal cancer that are not usually sensitive to these coordination complexes. Oxaliplatin has a non-hydrolysable diaminocyclohexane (DACH) carrier ligand which is maintained in the final cytotoxic metabolites of the drug. Like cisplatin, oxaliplatin targets DNA producing mainly 1,2-GG intrastrand cross-links. The cellular and molecular aspects of the mechanism of action of oxaliplatin have not yet been fully elucidated. However, the intrinsic chemical and steric characteristics of the DACH-platinum adducts appear to contribute to the lack of cross-resistance with cisplatin. To date, mismatch repair and replicative bypass appear to be the processes most likely involved in differentiating the molecular responses to these agents. Received 15 March 2002; received after revision 13 May 2002; accepted 21 May 2002 RID="*" ID="*"Corresponding author.     

Babesia bovis: the effect of acute inflammation and isoantibody production in the detection of babesial antigens

Summary Immunoblots ofBabesia bovis antigen contain dominant antigens which react not only with antisera toB. bovis but with sera from naive calves recovering from an acute inflammatory reaction. It seems likely these antigens are from the host rather than the parasite.     

NOD-like receptors: Ancient sentinels of the innate immune system

NOD-like receptors (NLRs) comprise a family of cytosolic proteins that have been implicated as ancient cellular sentinels mediating protective immune responses elicited by intracellular pathogens or endogenous danger signals. Genetic variants in NLR genes have been associated with complex chronic inflammatory barrier diseases (e.g. Crohn disease, bronchial asthma). In this review, we focus on the molecular pathophysiology of NLRs in the context of chronic inflammatory diseases and pinpoint recent advances in the evolutionary understanding of NLR biology. We propose that the field of NLRs may serve as a prototype for how a comprehensive understanding of an element of the immunological barrier will eventually lead to the development of targeted diagnostic, therapeutic and/or preventive strategies. Received 29 October 2007; received after revision 10 December 2007; accepted 19 December 2007     

The murine complement regulator Crry: new insights into the immunobiology of complement regulation

Complement has an important role in inflammation and in the normal function of the immune system. Activated complement fragments have the capacity to bind and damage self-tissues. Cells from vertebrates express on their surface regulators of complement activation that protect them from the deleterious effects of cell-bound complement fragments. Abnormalities in these regulators of complement activation may participate in the pathogenesis of autoimmune diseases and inflammatory disorders. Murine Crry is one of these regulators that inhibits the activation of the third component of complement and protects self-tissues from complement-mediated damage. Experimental work on Crry has increased our understanding of the immunobiology of complement regulation and the potential role of complement and complement inhibitors in the development and treatment of human diseases. Received 13 June 2001; received after revision 12 July 2001; accepted 9 August 2001     

Protein misfolding and disease: the case of prion disorders

Recent findings strongly support the hypothesis that diverse human disorders, including the most common neurodegenerative diseases, arise from misfolding and aggregation of an underlying protein. Despite the good evidence for the involvement of protein misfolding in disease pathogenesis, the mechanism by which protein conformational changes participate in the disease is still unclear. Among the best-studied diseases of this group are the transmissible spongiform encephalopathies or prion-related disorders, in which misfolding of the normal prion protein plays a key role in the disease. In this article we review recent data on the link between prion protein misfolding and the pathogensis of spongiform encephalopathies. Received 15 July 2002; received after revision 19 August 2002; accepted 23 August 2002 RID="*" ID="*"Corresponding author.     

Biosynthesis of 1-aminocyclopropanecarboxylic acid: Steric course of the reaction at the C-4 position

Summary Under the action of the appropriate synthase from ripe tomatoes a 11 mixture of (3S, 4R)-[3,4-²H₂] and (3R, 4S)-[3,4-²H₂]-(2S)-adenosylmethionine is transformed into a 11 mixture of the two meso forms of [²H₂]-1-aminocyclopropanecarboxylic acid, a result which proves the operation of an inversion mechanism and which is consistent with direct nucleophilic displacement of the leaving group in the substrate.     

Action of juvenile hormone on the follicle cells ofRhodnius prolixus: Evidence for a novel regulatory mechanism involving protein kinase C

Summary Juvenile hormone (JH) is known to act on the membranes of the follicle cells ofRhodnius, activating a specific Na⁺, K⁺-ATPase. This leads to a decrease in volume of the cells and the appearance of spaces between them (patency). The addition of an inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), to the medium in vitro inhibits the action of JH on the follicle cells. PDBU (phorbol-12,13-dibutyrate) mimics the action of JH in vitro and the response of the follicle cells to, PDBU is blocked by ouabain. It is concluded that the activation of protein kinase C is a required step in the chain of events leading to activation of the JH-dependent ATPase and set in train by the binding of JH to the membrane.     

Effects of exogenous juvenile hormone I on the numbers and distribution of antennal and maxillary and labial palp sensilla of maleBlattella germanica (L.) (Dictyoptera: Blattellidae)

Summary Unlike in the female, application of exogenous juvenile hormone I to 6th-instar male nymphs does not result in the retention of nymphal characteristics on the antennae and maxillary and labial palps. JH-treatment differentially affects the numbers of sensilla on the 3 appendages of adult maleB. germanica.     

Occludin binds to the SH3-hinge-GuK unit of zonula occludens protein 1: potential mechanism of tight junction regulation

The interaction between tight junction proteins occludin and zona occludens protein 1 (ZO-1) was clarified. The sequence cc1 within the hinge region of ZO-1, connecting its SH3 and GuK domains, was identified as a new association site for the occludin C-terminus, core binding area GLRSSKRNLRKSR (mouse ZO-1^606-618). Occludin also bound to the sequence H2 within GuK, core area HKLRKNNH (ZO-1^759-766). In occludin, the binding core was ELSRLDKELDDYREESEEY (mouse occludin^455-473). Helicity of the sequences was suggested by circular dichroism. Because basic residues in ZO-1, acidic residues in occludin (underlined), coiled-coil helix-forming leucine heptad motifs (bold) in occludin and, probably, in cc1 were essential, we conclude that interactions were both helical and ionic. Moreover, the GuK domain bound other GuK molecules, suggesting oligomerization of ZO-1. Generally, the assumption is supported that the SH3-hinge-GuK region represents a functional and regulatory unit in ZO-1 forming a multiprotein tight junction complex with occludin.Received 12 January 2004; received after revision 23 February 2004; accepted 31 March 2004     

Isoforms of soluble alpha-tubulin in oocytes and brain of the frog (genus Rana): changes during oocyte maturation

Rana oocytes have previously been shown to contain much more soluble tubulin than does the brain, suggesting different assembly and disassembly dynamics of frog oocyte tubulin compared to that in brain. By using centrifugation, SDS-PAGE, two-dimensional gel electrophoresis and Western blots, probed with anti-α-tubulin monoclonal antibodies, polymorphic α-tubulins (isoforms) were compared in brains and follicle-enclosed oocytes of northern (Rana pipiens) and southern (R. berlandieri) frogs. Oocyte tubulin in both species had isoforms with greater ranges of isoelectric point (pI) than those of brain tubulins; in particular, the oocyte tubulin pIs ranged further into the acidic region of the isoelectric-focusing gels than corresponding brain tubulin. This difference may, in part, be responsible for the previously reported assembly differences between oocyte tubulin (undetectable assembly) and brain tubulin (high assembly). Isoforms of α-tubulin with relat ively acidic pI were more abundant in northern frog brain and oocyte soluble extracts than in analogous extracts from southern frogs. Furthermore, additional acidic α-tubulin isoforms were found in progesterone-treated oocytes (i.e., eggs), indicating increased heterogeneity of acidic a-tubulin isoforms during oocyte meiotic maturation. Among northern frog oocyte soluble components fractionated on Superose-6b columns, tubulin complexes with apparent molecular mass of about 1800 kDa were found to contain acidic α-tubulin isoforms while the putative oligomeric tubulins with an apparent molecular mass of about 250 kDa contained an additional relatively basic α-tubulin isoform. The acidic α-tubulin isoforms, therefore, are proposed to be associated with cold-adaptable cells of brain and oocytes, and may also be involved in stabilization of large soluble tubulin complexes in oocytes of the northern frog. Received 1 October 2002; accepted 9 October 2002 RID="*" ID="*"Corresponding author.     

K252a: A new blocker of the cell-cycle at G1 phase in a human hepatoma cell line

The administration of 200 nM K252a to HuH7 suppressed the proliferation of the cells almost completely. The uptake of [³H]thymidine was inhibited, and flow cytometry revealed only one peak at 2C on day 3 after treatment with 100 nM K252a. The expression of proto-oncogene c-myc was not reduced. Despite the blockage at G1, both the size of the cells and the amount of cell protein had increased by 4 times by day 3 after treatment with K252a, while the cells secreted albumin and -fetoprotein into the medium as usual. These results show that K252a can increase the cell size of HuH7 without losing its function by blocking the cell cycle at G1 phase.     

Sex pheromone blend of the codling moth,Cydia pomonella: Evidence for a behavioral role of dodecan-1-ol

Summary Pheromone glands and effluvia of the codling moth female containE-8, E-10-dodecadien-1-ol as main component, accompanied by its geometric isomers, the corresponding acetate and aldehyde (both in gland extracts only),E-9-dodecen-1-ol and saturated alcohols of 10 to 18 carbons. Dodecan-1-ol as the most abundant secondary component (about 30% of the female blend) acts in the wind tunnel by widening the dose range over which codling moth males are optimally attracted toE-8, E-10-dodecadien-1-ol.This research was supported by the Swiss National Science Foundation. We thank W. Riggenbach for supplying moths and T. Wildbolz, P. Charmillot and M. Tóth for conducting field trials.     

Towards an understanding of the role of glutamate in neurodegenerative disorders: energy metabolism and neuropathology

It is thought that impairment, of energy metabolism that results in deterioration of membrane function, leading to loss of the Mg²⁺ block on NMDA receptors, and allowing persistent activation of these receptors by glutamate, might be a cause of neuronal death in neurodegenerative disorders. Studies in rodents using mitochondrial respiratory chain toxins, such as aminooxyacetic acid, 1-methyl-4-phenylpyridinium, malonic acid and 3-nitropropionic acid, suggest that such processes may indeed be involved in neurotoxicity. Striatal and nigral degeneration induced by mitochondrial toxins in rodents resembles the neuropathology seen in humans suffering from Huntington's or Parkinson's disease, and can be prevented either by decortication or by NMDA receptor antagonists. Such experimental observations suggest that glutamate may be involved in neuronal death leading to neurodegenerative disorders in humans. If so, glutamate antagonists may offer a therapeutic approach for retarding the progression of these disabling disorders.     

Arresting the mitotic oscillator and the control of cell proliferation: insights from a cascade model for cdc2 kinase activation

We consider a minimal cascade model previously proposed¹¹ for the mitotic oscillator driving the embryonic cell division cycle. The model is based on a bicyclic phosphorylation-dephosphorylation cascade involving cyclin and cdc2 kinase. By constructing stability diagrams showing domains of periodic behavior as a function of the maximum rates of the kinases and phosphatases involved in the two cycles of the cascade, we investigate the role of these converter enzymes in the oscillatory mechanism. Oscillations occur when the balance of kinase and phosphatase rates in each cycle is in a range bounded by two critical values. The results suggest ways to arrest the mitotic oscillator by altering the maximum rates of the converter enzymes. These results bear on the control of cell proliferation.