F-box proteins everywhere

The ubiquitin proteasome system is a key regulator of many biological processes in all eukaryotes. This mechanism employs several types of enzymes, the most important of which are the ubiquitin E3 ligases that catalyse the attachment of polyubiquitin chains to target proteins for their subsequent degradation by the 26S proteasome. Among the E3 families, the SCF is the best understood; it consists of a multi-protein complex in which the F-box protein plays a crucial role by recruiting the target substrate. Strikingly, nearly 700 F-box proteins have been predicted in Arabidopsis, suggesting that plants have the capacity to assemble a multitude of SCF complexes, possibly controlling the stability of hundreds of substrates involved in a plethora of biological processes. Interestingly, viruses and even pathogenic bacteria have also found ways to hijack the plant SCF and to reprogram it for their own purposes.     

GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways

The epidermal growth factor receptor (EGFR) plays a key role in the regulation of important cellular processes under normal and pathophysiological conditions such as cancer. In human mammary carcinomas the EGFR is involved in regulating cell growth, survival, migration and metastasis and its activation correlates with the lack of response in hormone therapy. Here, we demonstrate in oestrogen receptor-positive and -negative human breast cancer cells and primary mammary epithelial cells a cross-communication between G protein-coupled receptors (GPCRs) and the EGFR. We present evidence that specific inhibition of ADAM15 or TACE blocks GPCR-induced and proHB-EGF-mediated EGFR tyrosine phosphorylation, downstream mitogenic signalling and cell migration. Notably, activation of the PI3K downstream mediator PKB/Akt by GPCR ligands involves the activity of sphingosine kinase (SPHK) and is independent of EGFR signal transactivation. We conclude that GPCR-induced chemotaxis of breast cancer cells is mediated by EGFR-dependent and -independent signalling pathways, with both parallel pathways having to act in concert to achieve a complete migratory response.     

The styryl dye FM1-43 suppresses odorant responses in a subset of olfactory neurons by blocking cyclic nucleotide-gated (CNG) channels

Many olfactory receptor neurons use a cAMP-dependent transduction mechanism to transduce odorants into depolarizations. This signaling cascade is characterized by a sequence of two currents: a cation current through cyclic nucleotide-gated channels followed by a chloride current through calcium-activated chloride channels. To date, it is not possible to interfere with these generator channels under physiological conditions with potent and specific blockers. In this study we identified the styryl dye FM1-43 as a potent blocker of native olfactory cyclic nucleotide-gated channels. Furthermore, we characterized this substance to stain olfactory receptor neurons that are endowed with cAMP-dependent transduction. This allows optical differentiation and pharmacological interference with olfactory receptor neurons at the level of the signal transduction.     

Terpenes from pittosporaceae

The monoterpenes of the fruits of Pittosporum resiniferum and P. undulatum have been identified. The essential oil of P. resiniferum contai     

Effect of alemtuzumab-based T-cell depletion on graft compositional change in vitro and immune reconstitution early after allogeneic stem cell transplantation

Background aimsTo reduce the risk of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT), T-cell depletion (TCD) of grafts can be performed by the addition of alemtuzumab (ALT) “to the bag” (in vitro) before transplantation. In this prospective study, the authors analyzed the effect of in vitro incubation with 20 mg ALT on the composition of grafts prior to graft infusion. Furthermore, the authors assessed whether graft composition at the moment of infusion was predictive for T-cell reconstitution and development of GVHD early after TCD alloSCT.MethodsSixty granulocyte colony-stimulating factor-mobilized stem cell grafts were obtained from ≥9/10 HLA-matched related and unrelated donors. The composition of the grafts was analyzed by flow cytometry before and after in vitro incubation with ALT. T-cell reconstitution and incidence of severe GVHD were monitored until 12 weeks after transplantation.ResultsIn vitro incubation of grafts with 20 mg ALT resulted in an initial median depletion efficiency of T-cell receptor (TCR) α/β T cells of 96.7% (range, 63.5–99.8%), followed by subsequent depletion in vivo. Graft volumes and absolute leukocyte counts of grafts before the addition of ALT were not predictive for the efficiency of TCR α/β T-cell depletion. CD4^pos T cells were depleted more efficiently than CD8^pos T cells, and naive and regulatory T cells were depleted more efficiently than memory and effector T cells. This differential depletion of T-cell subsets was in line with their reported differential CD52 expression. In vitro depletion efficiencies and absolute numbers of (naive) TCR α/β T cells in the grafts after ALT incubation were not predictive for T-cell reconstitution or development of GVHD post- alloSCT.ConclusionsThe addition of ALT to the bag is an easy, fast and generally applicable strategy to prevent GVHD in patients receiving alloSCT after myeloablative or non-myeloablative conditioning because of the efficient differential depletion of donor-derived lymphocytes and T cells.     

Modulatory Effects of the Piccolo Genotype on Emotional Memory in Health and Depression

Major depressive disorder (MDD) has been associated with biased memory formation for mood-congruent information, which may be related to altered monoamine levels. The piccolo (PCLO) gene, involved in monoaminergic neurotransmission, has previously been linked to depression in a genome-wide association study. Here, we investigated the role of the PCLO risk allele on functional magnetic resonance imaging (MRI) correlates of emotional memory in a sample of 89 MDD patients (64 PCLO risk allele carriers) and 29 healthy controls (18 PCLO risk allele carriers). During negative word encoding, risk allele carriers showed significant lower activity relative to non-risk allele carriers in the insula, and trend-wise in the anterior cingulate cortex and inferior frontal gyrus. Moreover, depressed risk allele carriers showed significant lower activity relative to non-risk allele carriers in the striatum, an effect which was absent in healthy controls. Finally, amygdalar response during processing new positive words vs. known words was blunted in healthy PCLO+ carriers and in MDD patients irrespective of genotype, which may indicate that signalling of salient novel information does not occur to the same extent in PCLO+ carriers and MDD patients. The PCLO risk allele may increase vulnerability for MDD by modulating local brain function with regard to responsiveness to salient stimuli (i.e. insula) and processing novel negative information. Also, depression-specific effects of PCLO on dorsal striatal activation during negative word encoding and the absence of amygdalar salience signalling for novel positive information further suggest a role of PCLO in symptom maintenance in MDD.     

The Tissue Bank at the National Nuclear Research Institute in Mexico

The Instituto Nacional de Investigaciones Nucleares (ININ, The National Nuclear Research Institute) received during 1997-1998 strong support of the International Atomic Energy Agency (IAEA), to establish the first and only one tissue bank (BTR ININ tissue bank) in Mexico that uses ionising radiation as sterilising agent. In that time, the BTR staff was trained in different tissue banks in several countries. Basic equipment for tissue processing donated by the IAEA was received in 1998. In July, 1999 the Mexican Health Secretariat gave the Sanitary License No. 1062000001 to the BTR to operate as an official organ and tissue bank. In August, 2001 the ININ and the Hospital Materno Infantil (HMI-ISSEMYM) signed an agreement to collaborate in amnion processing. The hospital is responsible for donor selection, serology tests, tissue procurement and washing, since this hospital is the BTR amnion supplier. The tissues are collected by ININ weekly with complete documentation. The BTR is responsible for processing: cleaning, air drying, packaging, labelling, microbiological control and sterilisation by gamma irradiation. The sterilised tissue is kept under quarantine for 6 months to obtain the results of the donor second serology test. From March to June, 2002 the BTR has processed 347.86 units (50 cm(2) each), is say, 17,393 cm(2). In addition, the pig skin xenograft process has been implemented and a protocol for clinical applications of it is running at the Hospital Central Sur de Alta Especialidad (PEMEX). Also the ININ tissue bank present status and perspectives are described.     

Production of 5-methoxypodophyllotoxin in cell suspension cultures of Linum flavum L.

Cell suspension cultures of Linum flavum L., routinely grown on a NAA-containing medium, accumulated low levels of the phenylpropanoid-derived lignan 5-methoxypodophyllotoxin (5-MPT), up to 0.004% on a dry weight basis. Feeding experiments with the precursor L-phenylalanine resulted in a 3–5-fold increase in 5-MPT levels, but caused the levels of PAL activity to fall. Treatment of the cultures with the elicitor Nigeran, either alone or in combination with phenylalanine, caused the 5-MPT production to cease, even though PAL activity was rapidly enhanced by these treatments. Transfer of the cultures to NAA-free medium resulted in a 40–50 fold higher level of 5-MPT accumulation, the PAL activity levels being lowered compared to the routinely grown cells. With these more differentiated cultures, phenylalanine feeding and elicitor treatment, both on its own and in combination with the precursor, had no effect on 5-MPT production, even though the PAL activity levels were higher than in the untreated cells. It can be concluded that in lignan-accumulating cultures of L. flavum, PAL activity is nearly always detectable and seems to show a reciprocal relationship with 5-MPT accumulation.Abbreviations 5-MPT 5-methoxypodophyllotoxin - PAL phenylalanine ammonia lyase (EC 4:3:1.5) - NAA naphthaleneacetic acid