ate CCR2 Some reports have suggested that CCL2 could be involved

ate CCR2. Some reports have suggested that CCL2 could be involved in the early stages of CCR2 protein down modulation, while Navitoclax supplier other studies indicate that the differentiation proc ess itself, is a major factor in the selective loss of CCR2 gene e pression. Numerous cytokines are known to be involved in monocyte activation and differentiation, among them M CSF and IFN. M CSF is a lin eage specific hematopoetic growth factor that stimulates monocyte differentiation. The c fms proto onco gene encodes a high affinity receptor for M CSF and it has been shown that THP 1 cells e press this protein and that it is up regulated during differentiation. How ever, cells stimulated with M CSF alone for 48 hours did not lose e pression of CCR2.

Conversely, IFN alone, which is constitutively e pressed by monocyte lineage cells and which promotes matura tion of monocytes to macrophages, did significantly reduce e pression of CCR2, although the cells did not become adherent and neither did they change their mor phology. Interestingly, IFN has been demonstrated to up regulate levels of M CSF in mono cytes during maturation and when both IFN and M CSF were added, THP 1 cells did become adherent, changed their morphology and selectively lost CCR2, but not CCR1 all of which are characteristics of the mono cyte differentiation phenotype. These results are in keep ing with the studies published by Tangirala and colleagues, who reported similar phenomena in THP 1 cells. In addition, our studies also demonstrated that the regulatory effects mediated by IFN plus M CSF occurred at the level of transcription, where a significant down regulation in CCR2 promoter activity was observed.

Moreover, in the presence of staurosporine, IFN plus M CSF was unable to down regulate levels of CCR2. This result probably reflects the fact that IFN signals e ten sively through the JAK STAT pathway, and studies have suggested that staurosporine can block phosphorylation Drug_discovery of Janus kinases. In addition, we have found two putative binding sites in the CCR2 promoter for STAT transcription factors which would further support the contention that these transcription factors may be impor tant in the regulation of IFN mediated downregulation of CCR2. Conclusion This study demonstrates that e pression of the chemokine receptor CCR2 is e quisitely correlated with monocyte maturation.

selleck chemicals Freshly isolated monocytes e press high lev els of both CCR2 RNA and protein, whereas monocyte derived macrophages e press neither CCR2 RNA nor pro tein. Conversely, levels of the closely related chemokine receptor CCR1 remained stable and elevated throughout monocyte maturation. An analysis of the biochemical and molecular mechanisms underlying the regulated e pres sion of CCR2 revealed the e istence of several signaling pathways that selectively down modulate CCR2 gene e pression during monocyte differentiation. this e pres sion was largely regulated at the level of transcription. Sig naling through PMA and IFN plus M CSF, b

Ca2, responses to either UTP or UDP were not abolished Neverthel

Ca2, responses to either UTP or UDP were not abolished. Nevertheless, ma imal responses generated by UTP averaged 366 13%, significantly less than those observed in normal selleck chemicals FTY720 Krebs solution. The EC50 obtained for UTP in Ca2 free solution was 6. 2 0. 9 uM and was not significantly different from that obtained in normal Krebs. For UDP, similar findings were observed the ma imal response reached 230 15% and had an EC50 of 4. 9 0. 6 uM. neither parameter differed significantly from that in normal Krebs. This suggested that e tracellular Ca2 was not the major source of the i increase produced in TIC by UTP or UDP. more probably, this increase came from intracellular reservoirs via IP3 synthesis, as shown in other cell systems.

UTP induced activation of p44 and p42 MAPK In order to study the signaling pathway involved in the UTP and UDP activation of P2Y receptors in TIC, phos phorylation of the p44 and p42 MAPK proteins was eval uated. For these e periments, UTP was used as a specific agonist of the P2Y receptor subtypes studied. It was observed that UTP induced MAPK phosphorylation in a dose dependent manner with an EC50 of 3. 3 0. 9 and 1. 4 0. 7 uM for p44 and p42, respectively. ma imal increases of 541 25. 6% and 461 34. 8%, respectively, were observed by applying 100 uM UTP. The time course of this effect was studied by applying 10 uM UTP and measuring p44 and p42 MAPK phosphoryla tion at different times. The results indicated that ma imal phosphorylation occurred at 5 min of stimulation, and then it decreased slowly, returning to near basal levels about 30 min after UTP addition.

Because it has been shown consistently that UDP acts more potently on P2Y6 receptors, its ability to promote p44 and p42 MAPK phosphorylation was tested. In e periments similar to those presented above for UTP, 100 uM UDP was less potent and induced only modest responses of 199 43% and 158 15% for p44 and p42, respectively, compared to the basal level. the effect increased to 364 63% and 349 95%, Cilengitide respectively, with 1 mM UDP. The time course of p44 p42 phosphorylation induced by 1 mM UDP was similar to that elicited by 100 uM UTP. In addition, the p44 and p42 MAPK phosphorylation induced by 10 uM UTP was antagonized by suramin with an IC50 of 84. 3 10. 2 uM, suramin is a potent antagonist of P2Y2 receptors but is only a weak antagonist of P2Y6.

Conversely, PPADS up to 600 uM, a drug that antag onizes mainly the P2Y6 receptor, had no effect on UTP induced MAPK phosphorylation. These results suggested selleck compound that P2Y6 is not a major participant in the phosphorylation of MAPK. in consequence, the fol lowing e periments focused on defining the role of the P2Y2 receptor in the purinergic response. UTP induced p44 and p42 MAPK phosphorylation is dependent on PKC activation UTP dependent p44 p42 MAPK phosphorylation might be elicited via either of two main mechanisms 1 transac tivation of EGF receptors as has been demonstrated, for e ample, in salivary gland cells or 2 by activation of

me resis tant after initial therapy, necessitating second and eve

me resis tant after initial therapy, necessitating second and even third line treatment therapies. Thus, there is a need for additional new anti cancer drugs that induce specific cell death pathways in leukemia cells. It has recently been shown that the HIV protease inhibitor nelfinavir can induce cell death in a variety of human cancer types, and clinical studies with nelfinavir are currently INCB028050 proposed or underway. Nelfinavir appears to induce cell death in human cancer cells by rather pleiotropic mechanisms, including apoptosis, necrosis, and autophagy. Swelling of the endoplas mic reticulum by an accumulation of misfolded proteins appears to be a central mechanism in nelfinavir induced death in several cancer types, including lung cancer, glioma, and ovarian cancer cells, and precedes the activation of apoptosis.

Apoptosis can be induced by several pathways, includ ing an e trinsic pathway mediated by cell membrane bound death receptors and an intrinsic pathway mediated by activation of pro apoptotic intracellular mechanisms. Mitochondria play a central role in the induction and control of apoptosis because they harbour several apoptosis inducing proteins within their mem branes that can be released into the cytosol to induce caspase dependent cell death. Release of these mitochondrial factors occurs via outer mitochondrial membrane pore forma tion by pro apoptotic bcl 2 family members, such as ba , bak and t bid. The activities of these pro apoptotic molecules are counterbalanced by the anti apoptotic mitochondrial membrane proteins bcl 2, bcl L, and mcl 1.

Although there are several different the ories regarding how the pro and anti apoptotic bcl 2 family members interact, it has repeatedly been shown and is generally believed that increased e pres sion of pro apoptotic bcl 2 family members promotes cell death, whereas increased e pression of anti apopto tic bcl 2 family members facilitates cell survival. The most prominent anti apoptotic bcl 2 family members, including bcl 2, bcl L and mcl 1, were originally identified and found to be over e pressed in leukemia cells. Mcl 1 is a rather unique member of the bcl 2 family in that it has a rela tively large molecular weight of 40 42 kDa, compared to the molecular weight of ca. 26 kDa common to most other bcl 2 family members. Mcl 1 is a target of several pro apoptotic proteins and has been shown to undergo caspase mediated degradation during apoptosis.

Further, a shorter splice form of mcl 1 has been described and has been shown to e ert a pro apoptotic function. Thus, e pression Drug_discovery and modifica tion of mcl 1 appears to be crucial for regulation of cell survival and cell death in leukemia cells. In the present study, we show that despite its ability to induce apoptosis, nelfinavir enhances e pression of the mito chondria protective selleck chemicals mcl 1 protein in leukemia cells, resulting in a primarily mitochondria independent cas pase activation and cell death. Methods Cells and cell culture The human leukemia

lance between biological replicates, greater differences between

lance between biological replicates, greater differences between the time points of 3 h and 48 h, and interesting discriminant signals ARQ197 NSCLC such as those related to LITAF and IAP like apoptosis inhibitors. The general AMP down regulation detected in the hemocytes of Vibrio injected mussels confirms previous qPCR data. Similarly, putative acute phase response proteins and the macro phage Migration Inhibitory Factor were under expressed. Conversely, probes pointing to Allo graft inflammatory factor 1, SOD, small HSP20, plasmi nogen as well as various recognition receptors and molecules supporting intracellular signalling or cytoskeleton remodelling motility were commonly up regulated. Compared to the early response, after 2 days we detected a significant expression of proteases and pro tease inhibitors, LITAF and sequences suggesting various cell functions.

In general, no consistent trends could be defined for the C1q like and lectin like molecules. Due to their abundance and high sequence diversity, further study is necessary to understand their constitutive and PAMP induced expression in mussel hemocytes. Based on the Immunochip hybridization data, the molecular pathways and gene functions mapping out the mussel hemocyte response to the Vibrio injection are modelled in Figure 5. Functionally similar to dendri tic cells or macrophages, the mussel hemocytes display a pleiotropic response to the bacterial attack. Interacting with bacterial PAMPs, versatile and redundant recogni tion receptors undergo conformational changes, oligo merization or clustering.

The subsequent activation of cross talking signal transduction pathways adjusts the biochemical cell machinery towards the expression of specific gene sets and key effector molecules. Pathogen induced oxidative burst and damage associated Batimastat molecular patterns also sustain the inflammasome activation and intracellu lar signalling. Eventually, the endolysosomal and proteasome systems, secretory pathways and whole cell behaviour are recruited to achieve the pathogen killing. Overview of the mussel response to live Vibrio splendidus The most ancient defences of the living organisms are based on neuropeptide and protein hormone receptors, receptor kinases and PRR able to signal the danger and increase the expression of various inflammatory and effector molecules.

In view of the most recently sequenced invertebrate genomes, the Erlotinib EGFR pleiotropic innate immune responses could be described as a coordinated system of elements rapidly evolving and expanding the ability for pathogen sensing targeting, and evolutionary conserved regulatory factors which finely adjust basic cell processes and direct the development and perfor mance of the immune cells. Ancient signalling pathways like those of MAPKs and NFkB are not exclusive of the immune responses and, not solved by standard sequence searching, the identifi cation of invertebrate interleukine homologues makes new exploratory approaches necessary. Although the hemolymph ce

etabolism genes, although effects were still relatively small A

etabolism genes, although effects were still relatively small. A noteworthy result was the down regulation of elovl2 in salmon presenting higher flesh lipid, independent of LC PUFA content. Elovl2 then has substrate specificity towards LC PUFA and is highly responsive to dietary n 3 LC PUFA levels in sal mon. However, the expression of this gene is often co ordinately regulated with other genes of LC PUFA biosynthesis, such as 5fad and 6fad, which was not the case here. Hence, the biological significance of this result is not clear and may indicate other roles of elovl2 in lipid metabolism. For instance, an association between overexpression of elovl2 and enhanced triacyl glycerol synthesis and lipid droplet accumulation, as well as induction of PPAR�� target genes, was shown in mouse preadipocyte cell lines.

In addition, elovl2 was up regulated in the liver transcriptome of rats with nephrotic syndrome, a condition characterized by hyper lipidemia. Elovl2 was only recently characterized in salmon, and this is the first indication of an associ ation between its expression and lipid accumulation in a non mammalian vertebrate, with results suggesting that increased lipid level in salmon flesh repressed elovl2 expression independent of n 3 LC PUFA level although this requires further investigation. Another gene down regulated at higher lipid levels was a mitochondrial acyl carrier protein, involved in acyl transfer steps, including roles in fatty acid synthesis and functioning of the elec tron transport chain, which could conceptually be responding to similar regulatory mechanisms affecting elovl2.

In contrast, stearoyl CoA desaturase, responsible for the synthesis of monounsaturated fatty acids from saturated precursors, was up regulated in salmon with higher flesh lipid levels. This gene was positively corre lated with fat accumulation in bovine skeletal muscle, consistent with up regulation in salmon families with increased fat stores. Possible association Carfilzomib between flesh n 3 LC PUFA contents and immune response The predominance of immune response genes responding to total lipid level and, particularly, n 3 LC PUFA con tents in salmon flesh was unexpected. This was a true over representation as GO enrichment analysis enabled identification of several GO terms related to regulation of immune and inflammatory responses in relation to the total lipid factor.

However, as mentioned above, the tran scriptomic comparison, although balanced for total lipid, was not balanced for viral disease resistance and, as a consequence, higher contrast between families was imposed on the high lipid selleck Calcitriol group due to the fortuitous selection of family HH presenting a much higher viral resistance EBV. Nonetheless, if family HH biased the results of the two way ANOVA we would expect a preponderance of immune related genes to occur only when comparing these two families, presenting higher and lower flesh n 3 LC PUFA contents at the higher lipid level. In order to assess this, t test

le culture conditions and physiological synchronization of replic

le culture conditions and physiological synchronization of replicate batch cultures. The use of minimal medium with maltose as the sole limiting nutri useful site ent, constant pH, su?cient aeration and homogeneously dispersed mycelial biomass reduced biological and tech nical variations to a minimum and allowed us to highlight those di?erences in gene expression, which were in direct relation to carbon starvation. Submerged growth is fundamentally di?erent from the natural fungal life style. Fungi experience spatio temporal gradients of various ambient factors such as nutrients, temperature and pH in their natural habitats. These gradi ents lead to heterogenity within the fungal colony. Several studies have investigated this heterogeneity during growth on agar plates and have characterized di?erential concen tric zones with respect to gene expression and protein secretion.

Recently, this heterogeneity has even been shown for microcolonies in liquid shaken cultures of A. niger. In an ideally mixed bioreactor, all dispersed hyphae experience identical environmental conditions and temporal pro?les can be monitored and controlled by process parameters. Accordingly, many evo lutionary acquired traits contributing to the natural fungal life style such as the formation of substrate exploring hyphae, secretion of certain hydrolases, cell death and conidiation are dispensable during industrial processes and might even negatively a?ect production yields. In this study A. niger showed general hallmarks of autol ysis during prolonged carbon starvation. However, in contrast to A. nidulans, A.

niger hyphae did not undergo substantial fragmentation. While an increasing number of hyphal compartments became empty after car bon depletion, microscopic analysis showed that hyphal cell wall skeletons remained mainly intact. Thus disinte gration of aging mycelia appears rather to be initiated by intracellular activities such as cell death and or endoge nous recycling of neighboring compartments leading to empty hyphal ghosts than by extracellular hydrolysis of fungal cell walls. This assumption is supported by studies in A. nidulans, where autolytic fragmentation of hyphae and cell death were described as simultaneous but independently regulated processes. While dele tion of the major carbon catabolite repressor CreA in A.

nidulans resulted in increased hydrolase activities and mycelial Drug_discovery fragmentation during carbon starvation, the via bility of selleck catalog A. nidulans was not a?ected. Consistently, we observed hyphal fragmentation and enhanced biomass decline in bioreactor cultures during the starvation phase only when the pH control was switched o? leading to an elevated pH of approximately 5. 8 towards the end of cultivation. We thus pro pose that hydrolytic weakening of the fungal cell wall and hyphal fragmentation is a secondary e?ect, which occurs after initial cell death events and only under favorable conditions. In ?ow chamber experiments with A. oryzae, Pollack et al. followed singl

The range of chemical transformations remains unparalleled in the

The range of chemical transformations remains unparalleled in the laboratory. With few noteworthy exceptions, chemists have primarily focused on mononuclear transition metal complexes in developing homogeneous catalysis. Our group is interested in the development of selleck inhibitor carbon-heteroatom bond-forming reactions, with a particular focus on identifying reactions that can be applied to the synthesis of complex molecules. In this context, we have hypothesized that bimetallic redox chemistry, in which two metals participate synergistically, may lower the activation barriers to redox transformations relevant to catalysis. In this Account, we discuss redox chemistry of binuclear Pd complexes and examine the role of binudear intermediates in Pd-catalyzed oxidation reactions.

Stoichiometric organometallic studies of the oxidation of binuclear Pd-II complexes to binuclear Pd-III complexes and subsequent C-X reductive elimination from the resulting binudear Pd-III complexes have confirmed the viability of C-X bond-forming reactions mediated by binuclear Pd-III complexes. Metal metal bond formation, which proceeds concurrently with oxidation of binuclear Pd-II complexes, can lower the activation barrier for oxidation. We also discuss experimental and theoretical work that suggests that C-X reductive elimination is also facilitated by redox cooperation of both metals during reductive elimination. The effect of ligand modification on the structure and reactivity of binuclear Pd-III complexes will be presented in light of the impact that ligand structure can exert on the structure and reactivity of binudear Pd-III complexes.

Historically, oxidation reactions similar to those discussed here have been proposed to proceed via mononudear Pd-IV intermediates, and the hypothesis of mononuclear Pd-II/IV catalysis has guided the successful development of many reactions. Herein we discuss differences between monometallic Pd-IV and bimetallic Pd-III redox catalysis. We address whether appreciation of the relevance of bimetallic Pd-III redox catalysis is of academic interest exclusively, serving to provide a more nuanced description of catalysis, or if the new insight regarding bimetallic Pd-III chemistry can be a platform to enable future reaction development. To this end, we describe an example in which the hypothesis of bimetallic redox chemistry guided reaction development, leading to the discovery of reactivity distinct from monometallic catalysts.

“Oxidation reactions are key transformations in organic chemistry because they can increase chemical complexity and incorporate heteroatom substituents into carbon-based molecules. This principle is manifested in the conversion GSK-3 of petrochemical they feedstocks into commodity chemicals and in the synthesis of fine chemicals, pharmaceuticals, and other complex organic molecules.

These proteins included known modifiers of tau proteotoxicity, su

These proteins included known modifiers of tau proteotoxicity, such as ILF-2 (NFAT), ILF-3, and ataxin-2. A striking observation from the data set selleckchem Ponatinib was that tau binding to heat shock protein 70 (Hsp70) decreased, whereas binding to Hsp90 significantly increased. Both chaperones have been linked to tau homeostasis, but their mechanisms have not been established. Using peptide arrays and binding assays, we found that Hsp70 and Hsp90 appeared to compete for binding to shared sites on tau. Further, the Hsp90-bound complex proved to be important in initiating tau clearance in cells. These results suggest that the relative levels of Hsp70 and Hsp90 may help determine whether tau is retained or degraded.

Consistent with this model, analysis of reported microarray expression data from Alzheimer’s disease patients and age-matched controls showed that the levels of Hsp90 are reduced in the diseased hippocampus. These studies suggest that Hsp70 and Hsp90 work together to coordinate tau homeostasis.
Secreted tyrosinase from melanin-forming Streptomyces avermitilis MA4680 was involved in both ortho-hydroxylation and further oxidation of trans-resveratrol, leading to the formation of melanin. This finding was confirmed by constructing deletion mutants of melC(2) and melD(2) encoding extracellular and intracellular tyrosinase, respectively; the melC2 deletion mutant did not produce piceatannol as well as melanin, whereas the melD2 deletion mutant oxidized resveratrol and synthesized melanin with the same yields, suggesting that MelC2 is responsible for ortho-hydroxylation of resveratrol.

Extracellular tyrosinase (MelC2) efficiently converted trans-resveratrol into piceatannol in the presence of either tyrosinase inhibitors or reducing agents such as catechol, NADH, and ascorbic acid. Reducing agents slow down the dioxygenase reaction of tyrosinase. In the presence of catechol, the regio-specific hydroxylation of trans-resveratrol was successfully performed by whole cell biotransformation, and further oxidation of trans-resveratrol was efficiently blocked. The yield of this ortho-hydroxylation of trans-resveratrol was dependent upon inhibitor concentration. Using 1.

8 Entinostat mg of wild-type Streptomyces avermitilis cells, the conversion yield of 100 mu M trans-resveratrol to piceatannol was 78% in 3 h in the presence Gemcitabine order of 1 mM catechol, indicating 14 mu M piceatannol h(-1) DCW mg(-1) specific productivity, which was a 14-fold increase in conversion yield compared to that without catechol, which is a remarkably higher reaction rate than that of P450 bioconversion. This method could be generally applied to biocatalysis of various dioxygenases.
Sustained treatment of prostate cancer with androgen receptor (AR) antagonists can evoke drug resistance, leading to castrate-resistant disease. Elevated activity of the AR is often associated with this highly aggressive disease state.

Natural and synthetic derivatives of benzo-gamma-pyrones (i e fl

Natural and synthetic derivatives of benzo-gamma-pyrones (i.e. flavones, chromones, and coumarins) and their synthetic analogues possess a wide range of biological properties in vitro and in vivo. In this paper we investigated the influence of two hydrazone compounds of chromones, 3-[(2-dimethoxytiophosphoryl)-2-methylhydrazono]-methyl-chromen-4-one (CH-3) and 2-amino-6-chloro-3-[(2-hydroxyethyl)-hydrazonomethyl]-chromen-4-one sellckchem (A-12), on lipid peroxidation and bFGF concentration in the HL-60 cells. Both of the studied compounds had a significant influence on bFGF and TBARS in ranges -137.20 similar to 380.26% and -81.66 similar to -28.68%, respectively, in comparison with the control (counted as 0 %).
Patients with acute promyelocytic leukemia (APL) are prone to both bleeding and thrombosis.

The bleeding complications are well known. In contrast, APL-associated thrombosis is relatively underappreciated. We aimed to explore the issue of APL-associated thrombosis events. In the past 20 years, 127 cases with APL were found in our hospital database. We collected their coagulation laboratory profiles, including leukemia burdens, white blood cell and platelet counts, prothrombin time, activated partial thromboplastin time, fibrinogen levels, and disseminated intravascular coagulation scores. Data were compared between patients with or without thrombosis. Clinical outcomes and potential risk factors were obtained for analysis. Ten cases with APL-associated thrombosis were found. The incidence of thrombosis was 7.9% in our cohort.

Five patients had cerebral infarction, 5 had catheter-related thrombosis and 1 had acute myocardial infarction. No laboratory data were associated with clinical thrombosis. Three patients died during the induction phase but thrombosis was not the direct cause of death for any of them. We conclude that patients with APL are susceptible to thrombosis Anacetrapib in addition to bleeding. Laboratory coagulation parameters did not predict thrombosis in our series. Ischennic stroke and catheter-related thrombosis were the most common events in our Taiwanese cohort. Such a thrombosis pattern is unique and worth further investigation. Copyright (C) 2013 S. Karger AG, Basel
Background: Induction therapy for multiple myeloma (MM) and remission status before high-dose treatment (HDT) have been shown to be prognostic factors for survival outcome, although the optimal induction therapy is yet to be defined. Methods: We conducted a retrospective analysis of the impact of induction therapy on survival outcome before and after HDT in MM exactly patients. The study included 236 consecutive patients who underwent HDT.

However, RNAi of ddb 1 did not cause obvious LET 23 signalling de

However, RNAi of ddb 1 did not cause obvious LET 23 signalling defects. This might be due to incomplete knockdown, or alternatively, CDT 2 and CUL 4 could act independently selleck compound of DDB 1 in this context. We also provided in vitro evidence that CDT 2 can associate with SEM 5 directly. CDT 2 and SEM 5 share two functions, they attenuate LET 23 signalling during vulva development and are required for receptor mediated endocytosis during oogenesis. Linking these two functions together, we hypothesise that the CUL 4 DDB 1 CDT 2 E3 ubiquitin ligase might interact with SEM 5 to affect LET 23 endocytosis and attenuation of the LET 23 signalling cascade. However, our studies do not rule out an effect through other signalling pathways involved in vulva development such as Wnt or Notch.

The rereplication defect and LET 23 signalling The rereplication defect caused by depletion of CDT 2 or CUL 4 has been previously characterised as well as the cell cycle arrest phenotype. AV-951 However, it is difficult to explain that these defects could cause exces sive LET 23 signalling during vulva development. Indeed, experiments using hydroxyurea to arrest the VPC cell cycle have shown that egl 17 expression remains restricted to P6. p. Therefore, a replication block after first division as in the case of cul 4 deletion mutants is unlikely to cause persistent expression of egl 17,cfp. Furthermore, we observed increased LET 23 sig nalling in cdt 2 RNAi animals, and an increase in vulval fate adoption in gap 1, cdt 2 animals, under con ditions where the cell cycle proceeds normally.

There fore, the role of CDT 2 in preventing rereplication is likely to be independent of its function in preventing excess LET 23 signalling. CDT 2 may attenuate LET 23 signalling as a component of the CUL 4 DDB 1 E3 ligase complex RNAi by feeding in C. elegans has significant false nega tive rate, but false positives are rare. Hence, the finding that a deletion of cul 4 can cause the same vul val phenotype as cdt 2 suggests that both CUL 4 and CDT 2 are novel attenua tors of LET 23 signalling. Since purification of the human CUL 4 DDB 1 E3 ligase complex by different groups has identified CDT 2 as the substrate recognition unit, it is likely that CUL 4 and CDT 2 function together in the process of LET 23 attenuation. Even though, this study cannot rule out that CUL 4 could act in parallel to attenuate LET 23 signalling.

SEM 5 and attenuation of signalling SEM 5, the GRB2 homologue, has two activities linked to Receptor Tyrosine Kinase signalling. It can act as a positive regulator of signalling by recruiting SOS 1, or act as a negative modulator by recruiting SLI 1, the CBL homologue. SLI 1 is an E3 ubiqui tin ligase that can associate with SEM 5 to target RTKs and promote lysosomal degradation.