A PDZ domain protein interacts with the C-terminal tail of the insulin-like growth factor-1 receptor but not with the insulin receptor.

In this study, we report on the isolation of a PDZ domain protein, here designated as IIP-1, insulin-like growth factor-1 (IGF-1) receptor-interacting protein-1, which binds to the IGF-1 receptor, but not to the related insulin receptor, and which is involved in the regulation of cell motility. The interaction between the IGF-1 receptor and IIP-1 as well as a splice variant IIP-1/p26 was demonstrated in the yeast two-hybrid system. Using co-precipitation experiments, we confirmed the interaction in transfected cells as well as in vitro. Analysis of deletion mutants indicates that the PDZ domain of IIP-1 mediates interaction with the C-terminal tail of the IGF-1 receptor (serine-threonine-cysteine). This finding demonstrates that the C terminus of the IGF-1 receptor acts as novel PDZ domain binding site. Immunofluorescence analysis revealed an overlapping localization of IIP-1 and the IGF-1 receptor in the breast cancer cell line MCF-7. A functional connection between IIP-1 and the IGF-1 receptor is further supported by the finding that the level of expression of IIP-1 and the IGF-1 receptor strongly correlates in different normal and cancer cells. Furthermore, overexpression of IIP-1 resulted in an attenuation of migration of MCF-7 cells, which is one of the biological activities mediated by the IGF-1 signaling system.     

Regulation of the VLA integrin-ligand interactions through the beta 1 subunit

Integrins from the very late activation antigen (VLA) subfamily are involved in cellular attachment to extracellular matrix (ECM) proteins and in intercellular adhesions. It is known that the interaction of integrin proteins with their ligands can be regulated during cellular activation. We have investigated the regulation of different VLA-mediated adhesive interactions through the common beta 1 chain. We have found that certain anti-beta 1 antibodies strongly enhance binding of myelomonocytic U-937 cells to fibronectin. This beta 1-mediated regulatory effect involved both VLA-4 and VLA-5 fibronectin receptors. Moreover, anti-beta 1 mAb also induced VLA-4-mediated binding to a recombinant soluble form of its endothelial cell ligand VCAM-1. Non-activated peripheral blood T lymphocytes, unable to mediate VLA-4 interactions with fibronectin or VCAM-1, acquired the ability to bind these ligands in the presence of anti-beta 1 mAb. The anti-beta 1-mediated changes in the affinities of beta 1 integrin for their ligands were comparable to those triggered by different lymphocyte activation agents such as anti-CD3 mAb or phorbol ester. Adhesion of melanoma cells to other ECM proteins such as laminin or collagen as well as that of alpha 2-transfected K-562 cells to collagen, was also strongly enhanced by anti-beta 1 mAb. These beta 1-mediated regulatory effects on different VLA-ligand interactions do not involve changes in cell surface membrane expression of different VLA heterodimers. The anti-beta 1-mediated functional effects required an active metabolism, cytoskeleton integrity and the existence of physiological levels of intracellular calcium as well as a functional Na+/H+ antiporter. Beta 1 antibodies not only increased cell attachment but also promoted spreading and cytoplasmic extension of endothelial cells on plates coated with either fibronectin, collagen, or laminin as well as induced the rapid appearance of microspikes in U-937 cells on fibronectin. Moreover, both beta 1 integrin and the cytoskeletal protein talin colocalized in the anti-beta 1 induced microspikes. These results emphasize the central role of the common beta 1 chain in regulating different adhesive functions mediated by VLA integrins as well as cellular morphology.     

Nsl1p is essential for the establishment of bipolarity and the localization of the Dam-Duo complex

We identified a physical complex consisting of Mtw1p, an established kinetochore protein, with Nnf1p, Nsl1p and Dsn1p and have demonstrated that Nnf1p, Nsl1p and Dsn1p localize to the Saccharomyces cerevisiae kinetochore. When challenged prior to metaphase, the temperature-sensitive mutants nsl1-16 and nsl1-42 as well as Nsl1p-depleted cells failed to establish a bipolar spindle-kinetochore interaction and executed monopolar segregation of sister chromatids. In contrast, an nsl1-16 defect could not be evoked after the establishment of bipolarity. The observed phenotype is characteristic of that of mutants with defects in the protein kinase Ipl1p or components of the Dam-Duo kinetochore complex. However nsl1 mutants did not exhibit a defect in microtubule-kinetochore untethering as the ipl1-321 mutant does. Instead, they exhibited a severe defect in the kinetochore localization of the Dam-Duo complex suggesting this to be the cause for the failure of nsl1 cells to establish bipolarity. Moreover the analysis of Nsl1p-depleted cells indicated that Nsl1p is required for the spindle checkpoint and kinetochore integrity.     

Caspase-1: is IL-1 just the tip of the ICEberg?

Caspase-1, formerly known as interleukin (IL)-1-converting enzyme is best established as the protease responsible for the processing of the key pro-inflammatory cytokine IL-1β from an inactive precursor to an active, secreted molecule. Thus, caspase-1 is regarded as a key mediator of inflammatory processes, and has become synonymous with inflammation. In addition to the processing of IL-1β, caspase-1 also executes a rapid programme of cell death, termed pyroptosis, in macrophages in response to intracellular bacteria. Pyroptosis is also regarded as a host response to remove the niche of the bacteria and to hasten their demise. These processes are generally accepted as the main roles of caspase-1. However, there is also a wealth of literature supporting a direct role for caspase-1 in non-infectious cell death processes. This is true in mammals, but also in non-mammalian vertebrates where caspase-1-dependent processing of IL-1β is absent because of the lack of appropriate caspase-1 cleavage sites. This literature is most prevalent in the brain where caspase-1 may directly regulate neuronal cell death in response to diverse insults. We attempt here to summarise the evidence for caspase-1 as a cell death enzyme and propose that, in addition to the processing of IL-1β, caspase-1 has an important and a conserved role as a cell death protease.     

Modeling the hazard of transition into the absorbing state in the illness-death model

The illness-death model is the simplest multistate model where the transition from the initial state 0 to the absorbing state 2 may involve an intermediate state 1 (e.g., disease relapse). The impact of the transition into state 1 on the subsequent transition hazard to state 2 enables insight to be gained into the disease evolution. The standard approach of analysis is modeling the transition hazards from 0 to 2 and from 1 to 2, including time to illness as a time-varying covariate and measuring time from origin even after transition into state 1. The hazard from 1 to 2 can be also modeled separately using only patients in state 1, measuring time from illness and including time to illness as a fixed covariate. A recently proposed approach is a model where time after the transition into state 1 is measured in both scales and time to illness is included as a time-varying covariate. Another possibility is a model where time after transition into state 1 is measured only from illness and time to illness is included as a fixed covariate. Through theoretical reasoning and simulation protocols, we discuss the use of these models and we develop a practical strategy aiming to (a) validate the properties of the illness-death process, (b) estimate the impact of time to illness on the hazard from state 1 to 2, and (c) quantify the impact that the transition into state 1 has on the hazard of the absorbing state. The strategy is also applied to a literature dataset on diabetes.     

Disturbance of the Duration in the Menstrual Cycle under the Influence of Tight Clothing

We investigated the influence of skin pressure by clothing on the duration of menstrual cycle with 33 young adult women. The average age was 19.9 ± 2.1 years (mean ± SD), stature 159.5 ± 5.6cm and body mass 50.9 ± 5.5kg. Thirty-three women participated as subjects. They wore their usual clothing including foundation garments, panty stocking, pants or skirt and T-shirt or blouse and cardigan for the first 4 months from December to March ('Tight 1'). For the second 4 months from April to July, the women wore loose clothes, i.e., they did not wear foundation garments at home. Skirt and jeans were worn loosely ('Loose'). For the last 4 months, from August to November, they wore their clothes as tightly as possible, compared to 'Tight 1' ('Tight 2'). Each participant marked the first day of the occurrence of menses in the pocket diary throughout the year. The main results were summarized as follows: 1) The average duration of the menstrual cycle was 44.2 ± 14.9 days (mean ±) in 'Tight 1', 30.4 ± 3.0 days in 'Loose' and 47.4 ± 22.7 days in 'Tight 2'. 2) The number of months when the menses did not occur was 38 for 'Tight 1', 6 for 'Loose' and 40 for 'Tight 2'. 3). The number of participants who had a duration of the menstrual cycles for more than 40 days, was 25 participants for 'Tight', 10 for 'Loose' and 29 for 'Tight'. It can be concluded that skin pressure by clothing could disturb the duration in the menstrual cycle.     

Dissimilarity in the channel intrinsic stability among the bacterial KcsA and the inwardly rectifying potassium channel ROMK1

In this study, we compared the channel intrinsic stability of the bacterial K⁺-channel KcsA and the inwardly rectifying potassium channel (Kir) ROMK1. ROMK1 was successfully cloned, expressed and purified from Saccharomyces cerevisae. By conventional gel electrophoresis, ROMK1 was detected in monomeric form running exclusively at ～45 kDa either in its oxidized or reduced form. By perfluoro-octanoic acid (PFO)-PAGE, the reduced ROMK1 was identified as tetrameric as well as oligomeric complex. However, in its oxidized form ROMK1 was exclusively detected in oligomeric form thus indicating the role of intrinsic cysteine residues and formation of disulfide bonds in stabilizing the oligomeric ROMK1. On the other hand, KcsA purified from E. coli was detected as an extremely stable tetramer both in its oxidized or reduced forms as determined by conventional or PFO-PAGE. Furthermore, in planar lipid bilayer ROMK1 exhibited prominent inward rectification, low single channel conductance and high channel open probability as compared to the KcsA channel which showed typically slight outward rectification and low open probability under similar conditions. Our experiments clearly indicate that KcsA and ROMK1 channels differ with regard to their intrinsic stability which might be related to their structural and functional differences.     

PGH1, the precursor for the anti-inflammatory prostaglandins of the 1-series, is a potent activator of the pro-inflammatory receptor CRTH2/DP2

Prostaglandin H(1) (PGH(1)) is the cyclo-oxygenase metabolite of dihomo-γ-linolenic acid (DGLA) and the precursor for the 1-series of prostaglandins which are often viewed as "anti-inflammatory". Herein we present evidence that PGH(1) is a potent activator of the pro-inflammatory PGD(2) receptor CRTH2, an attractive therapeutic target to treat allergic diseases such as asthma and atopic dermatitis. Non-invasive, real time dynamic mass redistribution analysis of living human CRTH2 transfectants and Ca(2+) flux studies reveal that PGH(1) activates CRTH2 as PGH(2), PGD(2) or PGD(1) do. The PGH(1) precursor DGLA and the other PGH(1) metabolites did not display such effect. PGH(1) specifically internalizes CRTH2 in stable CRTH2 transfectants as assessed by antibody feeding assays. Physiological relevance of CRTH2 ligation by PGH(1) is demonstrated in several primary human hematopoietic lineages, which endogenously express CRTH2: PGH(1) mediates migration of and Ca(2+) flux in Th2 lymphocytes, shape change of eosinophils, and their adhesion to human pulmonary microvascular endothelial cells under physiological flow conditions. All these effects are abrogated in the presence of the CRTH2 specific antagonist TM30089. Together, our results identify PGH(1) as an important lipid intermediate and novel CRTH2 agonist which may trigger CRTH2 activation in vivo in the absence of functional prostaglandin D synthase.     

The role of the ganglioside lipid moiety in the process of ganglioside-cell interactions.

The role of the ceramide moiety of gangliosides, together with the deriving aggregative properties of ganglioside in solution, in the process of ganglioside-cell interactions was studied. The natural GM1(stearoyl) and the synthetic GM1(acetyl), containing the stearoyl and acetyl groups as the acyl moiety, respectively, were used in binding experiments to rat cerebellar granule cells. Regardless of the cell culture conditions, such as the presence of absence of fetal calf serum, the association of GM1(acetyl) to the cells was much greater than that of GM1(stearoyl). GM1(acetyl) was present in the incubation medium as monomers. After incubation, a large part of the total GM1(acetyl) associated to cells, 76-93% depending on the experimental conditions, was removed by washing with protein solutions. The remaining associated ganglioside was not removed by repeating washing with protein solutions or trypsin treatments and was considered as a component of the membrane. The cell association of GM1(stearoyl), present in solution as monomers as well as micelles, could be classified as serum-labile, trypsin-labile and trypsin-stable. The trypsin-stable form of association, corresponding to the molecules stably inserted into the membrane, was proportionally higher, the proportions varying with increasing incubation time and decreasing ganglioside concentration. This form of association was particularly high when incubation was performed in the presence of fetal calf serum. Incubation experiments performed with a mixture of GM1(stearoyl) and GM1(acetyl) in a molar ratio which allowed their presence in the medium as monomers as well as mixed micelles, led to a ganglioside association suggesting that besides the aggregative properties of the molecule other ganglioside properties are involved in the ganglioside-cell interaction process.     

Molecular determinants involved in the allosteric control of agonist affinity in the GABAB receptor by the GABAB2 subunit

The gamma-aminobutyric acid type B (GABAB) receptor is an allosteric complex made of two subunits, GABAB1 (GB1) and GABAB2 (GB2). Both subunits are composed of an extracellular Venus flytrap domain (VFT) and a heptahelical domain (HD). GB1 binds GABA, and GB2 plays a major role in G-protein activation as well as in the high agonist affinity state of GB1. How agonist affinity in GB1 is regulated in the receptor remains unknown. Here, we demonstrate that GB2 VFT is a major molecular determinant involved in this control. We show that isolated versions of GB1 and GB2 VFTs in the absence of the HD and C-terminal tail can form hetero-oligomers as shown by time-resolved fluorescence resonance energy transfer (based on HTRF technology). GB2 VFT and its association with GB1 VFT controlled agonist affinity in GB1 in two ways. First, GB2 VFT exerted a direct action on GB1 VFT, as it slightly increased agonist affinity in isolated GB1 VFT. Second and most importantly, GB2 VFT prevented inhibitory interaction between the two main domains (VFT and HD) of GB1. According to this model, we propose that GB1 HD prevents the possible natural closure of GB1 VFT. In contrast, GB2 VFT facilitates this closure. Finally, such inhibitory contacts between HD and VFT in GB1 could be similar to those important to maintain the inactive state of the receptor.     

Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.     

Structural-functional organization of the par region of the ColN plasmid

In the 679 b.p. SalI-KpnI-fragment of the small colicinogenic plasmid Co1N, the par-region has been localized, functioning at the expense of resolution of plasmid DNA multimer forms. It has been shown that the replication process of the monomeric form of the recombinant plasmid containing the Co1N par-region do not result in formation of a considerable number of multimers. Gene xer A product is necessary for the functioning of the multimer resolution mechanism of Co1N as well as Co1E1. Nucleotide sequence analysis of the Co1N par-region revealed the presence of essential homology with the par-locus of plasmid Co1E1. Results obtained in this work and data from literature indicate that par-regions of the Co1E1-type plasmids possess considerable homology, function according to a similar mechanism and represent the universal stability module of multicopy colicinogenic plasmids.     

Role of the Mf1-1 pheromone precursor gene of the filamentous ascomycete Cryphonectria parasitica

Site-directed recombination was used to obtain a Cryphonectria parasitica strain carrying deletions at the Mf1-1 gene locus. Macroscopic features such as growth rate and conidia production were unaffected by Mf1-1 deletions, but, when a strain containing a complete deletion of Mf1-1 was used as spermatia it was male sterile. The same strain was fully competent as a female parent. Deletion of three of the seven putative pheromone peptide repeats within the gene had no effect on mating. Male fertility of the complete deletion strain was restored when an ectopic copy of the Mf1-1 gene was returned by transformation. Expression of the mating type specific pheromone precursor gene Mf1-1 was stimulated by growth in nutritionally poor liquid media. It was found that age and source of inoculum of liquid cultures influences pheromone precusor gene expression, i.e., conidia did not express Mf1-1 and cultures derived from conidia were significantly delayed in expression of this gene, as were cultures derived from young mycelium. Cultures inoculated with older hyphae, however, expressed Mf1-1 within 1 day after inoculation.