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In the Drosophila wing, the Nedd4 ubiquitin ligases (E3s), dNedd4 and Su(dx), are important negative regulators of Notch signaling; they ubiquitinate Notch, promoting its endocytosis and turnover. Here, we show that Drosophila Nedd4 family interacting protein (dNdfip) interacts with the Drosophila Nedd4-like E3s. dNdfip expression dramatically enhances dNedd4 and Su(dx)-mediated wing phenotypes and further disrupts Notch signaling. dNdfip colocalizes with Notch in wing imaginal discs and with the late endosomal marker Rab7 in cultured cells. In addition, dNdfip expression in the wing leads to ectopic Notch signaling. Supporting this, expression of dNdfip suppressed Notch+/− wing phenotype and knockdown of dNdfip enhanced the Notch+/− wing phenotype. The increase in Notch activity by dNdfip is ligand independent as dNdfip expression also suppressed deltex RNAi and Serrate+/− wing phenotypes. The opposing effects of dNdfip expression on Notch signaling and its late endosomal localization support a model whereby dNdfip promotes localization of Notch to the limiting membrane of late endosomes allowing for activation, similar to the model previously shown with ectopic Deltex expression. When dNedd4 or Su(dx) are also present, dNdfip promotes their activity in Notch ubiquitination and internalization to the lysosomal lumen for degradation. [ABSTRACT FROM AUTHOR]

Although chromosome translocations are well-documented recurrent events in hematological malignancies and soft tissue sarcomas, their significance in carcinomas is less clear. We report here the molecular characterization of the reciprocal translocation t(1;15)(p22;q22) in the prostate carcinoma cell line, LNCaP. The chromosome 1 breakpoint was localized to a single BAC clone, RP11-290M5, by sequential FISH analysis of clones selected from the NCBI chromosome 1 map. This was further refined to a 580-bp region by Southern blot analysis. A 2.85-kb fragment spanning the der(1) breakpoint was amplified by long-range inverse PCR. The breakpoint on chromosome 1 was shown to lie between the CYR61 and the DDAH1 genes with the der(1) junctional sequence linking the CYR61 gene to the TSPAN3 (TM4SF8) gene on chromosome 15. Confirmatory PCR and FISH mapping of the der(15) showed loss of chromosome material proximal to the breakpoint on chromosome 15, containing the PSTPIP1 and RCN2 genes. On the available evidence we conclude that this translocation does not result in an in-frame gene fusion. Comparative expressed sequence hybridization (CESH) and comparative genomic hybridization (CGH) analysis, showed relative down-regulation of gene expression surrounding the breakpoint, but no gross change in genomic copy number. Real-time quantitative RT-PCR for genes around the breakpoint supported the CESH data. Therefore, here we may have revealed a gene down-regulation mechanism associated with a chromosome translocation, either through small deletion at the breakpoint or through another means of chromosome domain related gene regulation. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The cell line U937, which has been used extensively for studies of myeloid differentiation, bears the t(10;11)(p13;q14) translocation which results in a fusion between the MLLT10 (myeloid/lymphoid or mixed-lineage leukemia [trithorax, Drosophila, homolog]; translocated to 10; alias AF10) gene and the Ap-3-like clathrin assembly protein, PICALM (Clathrin assembly lymphoid myeloid leukaemia). Apart from this translocation, very little is known about the other genetic alterations in this cell line that may represent significant events in disease progression. In this study, conventional G-banding, CGH and M-FISH have been used to characterise fully all of the cytogenetic alterations present in the U937 cell line. M-FISH analysis confirmed the presence of the t(10;11) and an apparently normal copy of both chromosomes 10 and 11. A t(1;5) translocation was observed as well as several unbalanced rearrangements. CGH detected amplifications resulting from duplications of 2q, 6p and 13q. These changes could result in fusion gene products involved in carcinogenesis or the positions of putative oncogenes and tumour suppressor genes. A good correlation between conventional G-banding, CGH and M-FISH was observed. Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The article discusses localization of brain acid-soluble protein 1 (BASP1) to human chromosome 5p15.1->p14, differential expression in human cancer cell lines as a result of alterations in gene dosage. BASP1 encodes a neuron enriched Ca(2+)-dependent calmodulin-binding protein of unknown function. An Expressed Sequence Tag (EST) corresponding to BASP1 was found by Northern analysis to be differentially expressed in a panel of human cancer cell lines with highest expression in the cervical adenocarcinoma cell line, HeLa 53.

The article discusses localization of B cell lymphoma 6, member B (zinc finger protein) gene (BCL6B) to human chromosome 17p13.1 by in situ hybridization. Rearrangement of the B cell lymphoma 6 (BCL6) gene on chromosome 3q27 is a recurrent translocation event in non- Hodgkin's lymphoma. The frequency of these rearrangements depend on the histology of the tumour and are found in approximately 10% and 30 % of Follicular and Diffuse large B cell lymphomas respectively. There are three regions of homology between the two proteins, the BTB domain at the NH2-terminal region, a perfectly conserved 17 aa sequence in the middle region and the zinc finger domain at the COOH-terminus.

Presents an abstract of the article "The Prospective Identification of Patients at Risk of Developing Secondary Disease During a Triple FISH Assay Prior to High Dose Therapy," by Lisa Cunningham, N. Foot, A. Dixon-Mclver, M. Cross, C. Evans, T. A. Lister, and D. Lillington.

Glucagon signaling is essential for maintaining normoglycemia in mammals. The arrestin fold superfamily of proteins controls the trafficking, turnover, and signaling of transmembrane receptors as well as other intracellular signaling functions. Further investigation is needed to understand the in vivo functions of the arrestin domain-containing 4 (ARRDC4) protein family member and whether it is involved in mammalian glucose metabolism. Here, we show that mice with a global deletion of the ARRDC4 protein have impaired glucagon responses and gluconeogenesis at a systemic and molecular level. Mice lacking ARRDC4 exhibited lower glucose levels after fasting and could not suppress gluconeogenesis at the refed state. We also show that ARRDC4 coimmunoprecipitates with the glucagon receptor, and ARRDC4 expression is suppressed by insulin. These results define ARRDC4 as a critical regulator of glucagon signaling and glucose homeostasis and reveal a novel intersection of insulin and glucagon pathways in the liver., Competing Interests: Conflict of interest R. T. L. is a cofounder, scientific advisory board member, and private equity holder of Elevian, Inc. R. T. L. is a member of the scientific advisory board of Revidia Therapeutics, Inc, and the laboratory receives research support from BlueRock Therapeutics. J. K. K. is a scientific advisor of Elevian, Inc. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Relapse remains the most common cause of treatment failure for patients with acute myeloid leukemia (AML) who undergo allogeneic stem cell transplantation (alloSCT), and carries a grave prognosis. Multiple studies have identified the presence of measurable residual disease (MRD) assessed by flow cytometry before alloSCT as a strong predictor of relapse, but it is not clear how these findings apply to patients who test positive in molecular MRD assays, which have far greater sensitivity. We analyzed pretransplant blood and bone marrow samples by reverse-transcription polymerase chain reaction in 107 patients with NPM1-mutant AML enrolled in the UK National Cancer Research Institute AML17 study. After a median follow-up of 4.9 years, patients with negative, low (<200 copies per 105ABL in the peripheral blood and <1000 copies in the bone marrow aspirate), and high levels of MRD had an estimated 2-year overall survival (2y-OS) of 83%, 63%, and 13%, respectively (P < .0001). Focusing on patients with low-level MRD before alloSCT, those with FLT3 internal tandem duplications(ITDs) had significantly poorer outcome (hazard ratio [HR], 6.14; P = .01). Combining these variables was highly prognostic, dividing patients into 2 groups with 2y-OS of 17% and 82% (HR, 13.2; P < .0001). T-depletion was associated with significantly reduced survival both in the entire cohort (2y-OS, 56% vs 96%; HR, 3.24; P = .0005) and in MRD-positive patients (2y-OS, 34% vs 100%; HR, 3.78; P = .003), but there was no significant effect of either conditioning regimen or donor source on outcome. Registered at ISRCTN (http://www.isrctn.com/ISRCTN55675535)., (© 2020 by The American Society of Hematology.)

Extracellular vesicles (EVs) are lipid-bilayered vesicles that are released by multiple cell types and contain nucleic acids and proteins. Very little is known about how the cargo is packaged into EVs. Ubiquitination of proteins is a key posttranslational modification that regulates protein stability and trafficking to subcellular compartments including EVs. Recently, arrestin-domain containing protein 1 (Arrdc1), an adaptor for the Nedd4 family of ubiquitin ligases, has been implicated in the release of ectosomes, a subtype of EV that buds from the plasma membrane. However, it is currently unknown whether Arrdc1 can regulate the release of exosomes, a class of EVs that are derived endocytically. Furthermore, it is unclear whether Arrdc1 can regulate the sorting of protein cargo into the EVs. Exosomes and ectosomes are isolated from mouse embryonic fibroblasts isolated from wild type and Arrdc1-deficient (Arrdc1 -/- ) mice. Nanoparticle tracking analysis-based EV quantitation shows that Arrdc1 regulates the release of both exosomes and ectosomes. Proteomic analysis highlights the change in protein cargo in EVs upon deletion of Arrdc1. Functional enrichment analysis reveals the enrichment of mitochondrial proteins in ectosomes, while proteins implicated in apoptotic cleavage of cell adhesion proteins and formation of cornified envelope are significantly depleted in exosomes upon knockout of Arrdc1., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Newly synthesized transmembrane proteins undergo a series of steps to ensure that only the required amount of correctly folded protein is localized to the membrane. The regulation of protein quality and its abundance at the membrane are often controlled by ubiquitination, a multistep enzymatic process that results in the attachment of ubiquitin, or chains of ubiquitin to the target protein. Protein ubiquitination acts as a signal for sorting, trafficking, and the removal of membrane proteins via endocytosis, a process through which multiple ubiquitin ligases are known to specifically regulate the functions of a number of ion channels, transporters, and signaling receptors. Endocytic removal of these proteins through ubiquitin-dependent endocytosis provides a way to rapidly downregulate the physiological outcomes, and defects in such controls are directly linked to human pathologies. Recent evidence suggests that ubiquitination is also involved in the shedding of membranes and associated proteins as extracellular vesicles, thereby not only controlling the cell surface levels of some membrane proteins, but also their potential transport to neighboring cells. In this review, we summarize the mechanisms and functions of ubiquitination of membrane proteins and provide specific examples of ubiquitin-dependent regulation of membrane proteins., (Copyright © 2017 the American Physiological Society.)

Context: Individuals with a history of anterior cruciate ligament reconstruction (ACLR) are at greater risk of reinjury and developing early-onset osteoarthritis due to persistent abnormal joint loading. Real-time clinical assessment tools may help identify patients experiencing abnormal movement patterns after ACLR., Objective: To compare performance on the Landing Error Scoring System (LESS) between participants with ACLR and uninjured control participants and to determine the relationship between LESS score and knee-extension strength in these participants., Design: Controlled laboratory study., Setting: Research laboratory., Patients or Other Participants: Forty-six recreationally active participants, consisting of 22 with ACLR (12 men, 10 women; age = 22.5 ± 5.0 years, height = 172.8 ± 7.2 cm, mass = 74.2 ± 15.6 kg, body mass index = 24.6 ± 4.0) and 24 healthy control participants (12 men, 12 women; age = 21.7 ± 3.6 years, height = 168.0 ± 8.8 cm, mass = 69.2 ± 13.6 kg, body mass index = 24.3 ± 3.2) were enrolled., Main Outcome Measure(s): Bilateral normalized knee-extension maximal voluntary isometric contraction (MVIC) torque (Nm/kg) and LESS scores were measured during a single testing session. We compared LESS scores between groups using a Mann-Whitney U test and the relationships between LESS scores and normalized knee-extension MVIC torque using Spearman ρ bivariate correlations., Results: The ACLR participants had a greater number of LESS errors (6.0 ± 3.6) than healthy control participants (2.8 ± 2.2; t44 = -3.73, P = .002). In ACLR participants, lower normalized knee-extension MVIC torque in the injured limb (ρ = -0.455, P = .03) was associated with a greater number of landing errors., Conclusions: Participants with ACLR displayed more errors while landing. The occurrence of landing errors was negatively correlated with knee-extension strength, suggesting that weaker participants had more landing errors. Persistent quadriceps weakness commonly associated with ACLR may be related to a reduced quality of lower extremity movement during dynamic tasks.

In recent years it has become increasingly evident that MYC rearrangements are not confined to classical Burkitt lymphoma (BL), but also occur in diffuse large B-cell lymphoma (DLBCL) and in the new subtype, "B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL" (BCLU), which was recently described in the 2008 revision of the World Health Organization classification. The accurate identification of MYC rearrangements in these three subtypes of high-grade lymphoma is becoming increasingly critical both in terms of diagnosis of classical BL and in light of the prognostic implications in cases of DLBCL and BCLU. We describe three cases of high-grade lymphoma in which cryptic insertion events, resulting in clinically significant IGH-MYC rearrangements, were detectable using an IGH/MYC three-color, dual-fusion fluorescence in situ hybridization (FISH) probe set, but were not detected using break-apart MYC FISH probes, thus highlighting the limitations of using break-apart probes as a stand-alone test, particularly with the increased use of interphase FISH analysis of formalin-fixed, paraffin-embedded tissue sections in the diagnostic work-up of these patients., (Copyright 2010 Elsevier Inc. All rights reserved.)

This multicenter phase I/II study investigated the maximum tolerated dose (MTD), safety, and efficacy of low dose intravenous (IV) melphalan in combination with bortezomib for patients with relapsed multiple myeloma (MM). Patients received bortezomib 1.3 mg/m(2) on days 1, 4, 8, and 11 and escalating doses of IV melphalan (2.5-10.0 mg/m(2)) on day 2 of a 28-day cycle for a maximum of eight cycles. Dexamethasone 20 mg was added for progressive or stable disease. Fifty-three patients were enrolled. The MTD was defined at melphalan 7.5 mg/m(2) and bortezomib 1.3 mg/m(2). The overall response rate (ORR) was 68% (23% complete or near-complete responses [CR/nCR]) whilst at the MTD (n = 33) the ORR was 76% (34% CR/nCR). After median follow-up of 17 months, the median progression free survival was 10 months, rising to 12 months at the MTD (P < 0.05 vs. non-MTD regimens). The median overall survival was 28 months, but was not yet reached at the MTD. Grade 3/4 adverse events included thrombocytopenia (62%), neutropenia (57%), infection (21%), and neuropathy (15%). Bortezomib and low-dose IV melphalan combination therapy is a safe and highly effective regimen for patients with relapsed MM. These data suggest further investigation of this combination is warranted.

The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50-90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury, it is possible that exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress.

Bortezomib, doxorubicin and dexamethasone (PAD) was evaluated as induction before stem cell transplantation in newly diagnosed multiple myeloma (MM) patients, using bortezomib 1.3 mg/m(2) (PAD1, N = 21) or 1.0 mg/m(2) (PAD2, N = 20). Complete/very good partial response rates with PAD1/PAD2 were 62%/42% postinduction and 81%/53% post-transplant. Progression-free survival (29 vs. 24 months), time to re-treatment (36 vs. 29 months) and overall survival (1 year: 100% vs. 95%; 2 years: 95% vs. 73%) were statistically similar but favoured PAD1 versus PAD2. Toxicity was lower in PAD2; bortezomib dose reduction may help manage toxicities while retaining efficacy. PAD is highly active as front-line induction in MM.

Ubiquitination is essential in mediating diverse cellular functions including protein degradation and trafficking. Ubiquitin-protein (E3) ligases determine the substrate specificity of the ubiquitination process. The Nedd4 family of E3 ligases is an evolutionarily conserved family of proteins required for the ubiquitination of a large number of cellular targets. As a result, this family regulates a wide variety of cellular processes including transcription, stability and trafficking of plasma membrane proteins, and the degradation of misfolded proteins. The modular architecture of the proteins, comprising a C2 domain, multiple WW domains and a catalytic domain, enables diverse intermolecular interactions and recruitment to various subcellular locations. The WW domains commonly mediate interaction with substrate proteins; however, an increasing number of Nedd4 targets do not contain obvious WW domain-interaction motifs suggesting the involvement of accessory proteins. This review discusses recent insights into how accessory and adaptor proteins modulate the activities of Nedd4 family members, including recruitment of novel substrates, alteration of subcellular localisation and effects on ubiquitination.

We have prospectively analysed and correlated the gene expression profiles of children presenting with acute leukaemia to the Royal London and Great Ormond Street Hospitals with morphological diagnosis, immunophenotype and karyotype. Total RNA extracted from freshly sorted blast cells was obtained from 84 lymphoblastic [acute lymphoblastic leukaemia (ALL)], 20 myeloid [acute myeloid leukaemia (AML)] and three unclassified acute leukaemias and hybridised to the high density Affymetrix U133A oligonucleotide array. Analysis of variance and significance analysis of microarrays was used to identify discriminatory genes. A novel 50-gene set accurately identified all patients with ALL and AML and predicted for a diagnosis of AML in three patients with unclassified acute leukaemia. A unique gene set was derived for each of eight subtypes of acute leukaemia within our data set. A common profile for children with ALL with an ETV6-RUNX1 fusion, amplification or deletion of ETV6, amplification of RUNX1 or hyperdiploidy with an additional chromosome 21 was identified. This suggests that these rearrangements share a commonality in biological pathways that maintains the leukaemic state. The gene TERF2 was most highly expressed in this group of patients. Our analyses demonstrate that not only is microarray analysis the single most effective tool for the diagnosis of acute leukaemias of childhood but it has the ability to identify unique biological pathways. To further evaluate its prognostic value it needs to be incorporated into the routine diagnostic analysis for large-scale clinical trials in childhood acute leukaemias.

Comparative expression analysis of wild-typeETV6 in the disease state showed an absence of expression in ETV6-CBFA2 acute lymphoblastic leukaemia (ALL) when compared with non-ETV6-CBFA2 ALL and acute myeloid leukaemia. Fluorescent in-situ hybridization and loss of heterozygosity studies showed that 73% of the ETV6-CBFA2 samples had a fully or partially deleted second ETV6 allele, explaining the lack of wild-type expression in these patients. Although the second ETV6 allele was identified in the remaining patients, no ETV6 expression was detected. These observations support the hypothesis that loss of ETV6 expression is a critical secondary event for leukaemogenesis in ETV6-CBFA2 ALL.