65 research outputs found
Retrotransposition and mutation events yield Rap1 GTPases with differential signalling capacity
<p>Abstract</p> <p>Background</p> <p>Retrotransposition of mRNA transcripts gives occasionally rise to functional retrogenes. Through acquiring tempero-spatial expression patterns distinct from their parental genes and/or functional mutations in their coding sequences, such retrogenes may in principle reshape signalling networks.</p> <p>Results</p> <p>Here we present evidence for such a scenario, involving retrogenes of Rap1 belonging to the Ras family of small GTPases. We identified two murine and one human-specific retrogene of Rap1A and Rap1B, which encode proteins that differ by only a few amino acids from their parental Rap1 proteins. Markedly, human hRap1B-retro and mouse mRap1A-retro1 acquired mutations in the 12<sup>th </sup>and 59<sup>th </sup>amino acids, respectively, corresponding to residues mutated in constitutively active oncogenic Ras proteins. Statistical and structural analyses support a functional evolution scenario, where Rap1 isoforms of retrogenic origin are functionally distinct from their parental proteins. Indeed, all retrogene-encoded GTPases have an increased GTP/GDP binding ratio <it>in vivo</it>, indicating that their conformations resemble that of active GTP-bound Rap1. We furthermore demonstrate that these three Rap1 isoforms exhibit distinct affinities for the Ras-binding domain of RalGDS. Finally, when tested for their capacity to induce key cellular processes like integrin-mediated cell adhesion or cell spreading, marked differences are seen.</p> <p>Conclusions</p> <p>Together, these data lend strong support for an evolution scenario, where retrotransposition and subsequent mutation events generated species-specific Rap1 isoforms with differential signaling potential. Expression of the constitutively active human Rap1B-retro in cells like those derived from Ramos Burkitt's lymphoma and bone marrow from a patient with myelodysplastic syndrome (MDS) warrants further investigation into its role in disease development.</p
Determination of Ras-GTP and Ras-GDP in patients with acute myelogenous leukemia (AML), myeloproliferative syndrome (MPS), juvenile myelomonocytic leukemia (JMML), acute lymphocytic leukemia (ALL), and malignant lymphoma: assessment of mutational and indirect activation
The 21-kD protein Ras of the low-molecular-weight GTP-binding (LMWG) family plays an important role in transduction of extracellular signals. Ras functions as a âmolecular switchâ in transduction of signals from the membrane receptors of many growth factors, cytokines, and other second messengers to the cell nucleus. Numerous studies have shown that in multiple malignant tumors and hematopoietic malignancies, faulty signal transduction via the Ras pathway plays a key role in tumorigenesis. In this work, a non-radioactive assay was used to quantify Ras activity in hematologic malignancies. Ras activation was measured in six different cell lines and 24 patient samples, and sequence analysis of N- and K-ras was performed. The 24 patient samples comprised of seven acute myelogenous leukemia (AML) samples, five acute lymphocytic leukemia (ALL) samples, four myeloproliferative disease (MPD) samples, four lymphoma samples, four juvenile myelomonocytic leukemia (JMML) samples, and WBC from a healthy donor. The purpose of this study was to compare Ras activity determined by percentage of Ras-GTP with the mutational status of the Ras gene in the hematopoietic cells of the patients. Mutation analysis revealed ras mutations in two of the seven AML samples, one in codon 12 and one in codon 61; ras mutations were also found in two of the four JMML samples, and in one of the four lymphoma samples (codon 12). We found a mean Ras activation of 23.1% in cell lines with known constitutively activating ras mutations, which was significantly different from cell lines with ras wildtype sequence (Ras activation of 4.8%). Two of the five activating ras mutations in the patient samples correlated with increased Ras activation. In the other three samples, Ras was probably activated through âupstreamâ or âdownstreamâ mechanisms
Mammalian mitochondrial nitric oxide synthase: Characterization of a novel candidate
AbstractRecently a novel family of putative nitric oxide synthases, with AtNOS1, the plant member implicated in NO production, has been described. Here we present experimental evidence that a mammalian ortholog of AtNOS1 protein functions in the cellular context of mitochondria. The expression data suggest that a candidate for mammalian mitochondrial nitric oxide synthase contributes to multiple physiological processes during embryogenesis, which may include roles in liver haematopoesis and bone development
Rap Signaling in Normal Lymphocyte Development and Leukemia Genesis
Although Rap GTPases of the Ras family remained enigmatic for years, extensive studies in this decade have revealed diverse functions of Rap signaling in the control of cell proliferation, differentiation, survival, adhesion, and movement. With the use of gene-engineered mice, we have uncovered essential roles of endogenous Rap signaling in normal lymphocyte development of both T- and B-lineage cells. Deregulation of Rap signaling, on the other hand, results in the development of characteristic leukemia in manners highly dependent on the contexts of cell lineages. These results highlight crucial roles of Rap signaling in the physiology and pathology of lymphocyte development
PBX1 is dispensable for neural commitment of RA-treated murine ES cells
Experimentation with PBX1 knockout mice has shown that PBX1 is necessary for early embryogenesis. Despite broad insight into PBX1 function, little is known about the underlying target gene regulation. Utilizing the CreâloxP system, we targeted a functionally important part of the homeodomain of PBX1 through homozygous deletion of exon-6 and flanking intronic regions leading to exon 7 skipping in embryonic stem (ES) cells. We induced in vitro differentiation of wild-type and PBX1 mutant ES cells by aggregation and retinoic acid (RA) treatment and compared their profiles of gene expression at the ninth day post-reattachment to adhesive media. Our results indicate that PBX1 interactions with HOX proteins and DNA are dispensable for RA-induced ability of ES to express neural genes and point to a possible involvement of PBX1 in the regulation of imprinted genes
HPK1 Associates with SKAP-HOM to Negatively Regulate Rap1-Mediated B-Lymphocyte Adhesion
BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-related serine/threonine kinase activated by a range of environmental stimuli including genotoxic stress, growth factors, inflammatory cytokines and antigen receptor triggering. Being inducibly recruited to membrane-proximal signalling scaffolds to regulate NFAT, AP-1 and NFkappaB-mediated gene transcription in T-cells, the function of HPK1 in B-cells to date remains rather ill-defined. METHODOLOGY/PRINCIPAL FINDINGS: By using two loss of function models, we show that HPK1 displays a novel function in regulating B-cell integrin activity. Wehi 231 lymphoma cells lacking HPK1 after shRNA mediated knockdown exhibit increased basic activation levels of Ras-related protein 1 (Rap1), accompanied by a severe lymphocyte function-associated antigen-1 (LFA-1) dependent homotypic aggregation and increased adhesion to intercellular adhesion molecule 1 (ICAM-1). The observed phenotype of enhanced integrin activity is caused downstream of Src, by a signalling module independent of PI3K and PLC, involving HPK1, SKAP55 homologue (SKAP-HOM) and Rap1-GTP-interacting adaptor molecule (RIAM). This alters actin dynamics and renders focal adhesion kinase (FAK) constitutively phosphorylated. Bone marrow and splenic B-cell development of HPK1(-/-) mice are largely unaffected, except age-related tendencies for increased splenic cellularity and BCR downregulation. In addition, naĂŻve splenic knockout B-cells appear hyperresponsive to a range of stimuli applied ex vivo as recently demonstrated by others for T-cells. CONCLUSIONS/SIGNIFICANCE: We therefore conclude that HPK1 exhibits a dual function in B-cells by negatively regulating integrin activity and controlling cellular activation, which makes it an interesting candidate to study in pathological settings like autoimmunity and cancer
FPGA-accelererad paketfÄngst med eBPF : PrestandaövervÀganden vid anvÀndning av SoC FPGA acceleratorer för paketering.
With the rise of the Internet of Things and the proliferation of embedded devices equipped with an accelerator arose a need for efficient resource utilization. Hardware acceleration is a complex topic that requires specialized domain knowledge about the platform and different trade-offs that have to be made, especially in the area of power consumption. Efficient work offloading strives to reduce or at least maintain the total power consumption of the system. Offloading packet capturing is usually done in more powerful devices, hence scarce research is present concerning network packet acceleration in embedded devices. The thesis focuses on accelerating networking packets utilizing a Field Programmable Gate Array in an embedded Linux System. The solution is based on a custom Linux distribution assembled using the Buildroot tool, specially configured and patched Linux kernel, uboot bootloader, and the programmable logic for packet acceleration. The system is evaluated on a De0-Nano System on Chip development board through modifications to burst lengths, packet sizes, and programmable logic clock frequency. Metrics include packet capturing time, time per packet, and consumed power. Finally, the results are contrasted with baseline embedded Linux packet processing by inspection of a packetâs path through the kernel. Collected results provide a deeper understanding of the packet acceleration problem in embedded devices and the resultant system gives a solid starting point for possible extensions such as packet filtering. Key findings include an improvement in packet processing speed as the clock frequency and burst length are increased while maintaining power consumption. Additionally, the solution performs better when the packet sizes are above 64 bytes as the overhead of additional logic necessary for their processing is compensated. The project is also found to be significantly faster than regular in kernel processing with the caveat of providing just packet capturing whereas Linux contains a full network stack.I och med uppkomsten av sakernas internet och spridningen av inbyggda enheter som Ă€r utrustade med en accelerator har det uppstĂ„tt ett behov av effektivt resursutnyttjande. HĂ„rdvaruacceleration Ă€r ett komplext Ă€mne som krĂ€ver specialiserad domĂ€nkunskap om plattformen och olika avvĂ€gningar som mĂ„ste göras, sĂ€rskilt nĂ€r det gĂ€ller energiförbrukning. Effektiv arbetsavlastning strĂ€var efter att minska eller Ă„tminstone bibehĂ„lla systemets totala energiförbrukning. Avlastning av paketering sker vanligtvis i kraftfullare enheter, och dĂ€rför finns det knappt nĂ„gon forskning om nĂ€tverksacceleration av paket i inbyggda enheter. Avhandlingen Ă€r inriktad pĂ„ att pĂ„skynda nĂ€tverkspaket med hjĂ€lp av en Field Programmable Gate Array i ett inbĂ€ddat Linuxsystem. Lösningen bygger pĂ„ en anpassad Linuxdistribution som sammanstĂ€llts med hjĂ€lp av verktyget Buildroot, en sĂ€rskilt konfigurerad och patchad LinuxkĂ€rna, uboot bootloader och den programmerbara logiken för paketacceleration. Systemet utvĂ€rderas pĂ„ ett De0-Nano System on Chip-utvecklingskort genom Ă€ndringar av burstlĂ€ngder, paketstorlekar och den programmerbara logikens klockfrekvens. Metrikerna omfattar tid för paketering, tid per paket och förbrukad effekt. Slutligen jĂ€mförs resultaten med grundlĂ€ggande inbĂ€ddad Linux-paketbehandling genom inspektion av paketens vĂ€g genom kĂ€rnan. De samlade resultaten ger en djupare förstĂ„else för problemet med paketacceleration i inbyggda enheter och det resulterande systemet ger en solid utgĂ„ngspunkt för möjliga utvidgningar, t.ex. paketfiltrering. Bland de viktigaste resultaten kan nĂ€mnas en förbĂ€ttring av hastigheten i paketbehandlingen nĂ€r klockfrekvensen och burstlĂ€ngden ökas samtidigt som strömförbrukningen bibehĂ„lls. Dessutom fungerar lösningen bĂ€ttre nĂ€r paketstorleken Ă€r större Ă€n 64 bytes eftersom den extra logik som krĂ€vs för att behandla paketen kompenseras. Projektet har ocksĂ„ visat sig vara betydligt snabbare Ă€n vanlig kĂ€rnbearbetning, med den reservationen att det bara tillhandahĂ„ller paketupptagning, medan Linux innehĂ„ller en fullstĂ€ndig nĂ€tverksstack.RozwĂłj Internetu Rzeczy i Ä
rosnca ĆÄpopularno systemĂłw wbudowanych Ä
posiadajcych wbudowany akcelerator Äsprztowy Ćsprawiy, ĆŒe Ćwzrosa potrzeba na ich efektywne wykorzytanie. Akceleracja Äsprztowa jest Ä
dziedzin nauki, ktĂłra wymaga specjalistycznej wiedzy na temat platformy na ktĂłrej ma Äoperowa oraz wymaga Ćznajomoci potencjalnych komplikacji ktĂłre Äsi z Ä
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ĆŒÄ
wi. Efektywna akceleracja ma na celu Äredukcj ĆŒzuycia energii, a przynajmnniej jej utrzymanie na dotychczasowym poziomie. Tematyka ta jest ĆÄdo uboga pod Ä
ktem Ädostpnej literatury, ĆŒgdy zazwyczaj akceleratory stosowane do sieciowych Ä
Ćrozwiza Ä
s ĆŒuywane w Ä
rozwizaniach serwerowych gdzie ÄÄ
wystpuj innego rodzaju problemy. W pracy wykorzystany jest akcelerator Field Programmable Gate Array ktĂłry jest ÄĆÄ
czci Ćpytki deweloperskiej De0-Nano System on Chip, gdzie Ćdziaa ĆÄ
wspĂłpracujc z wbudowanym systemem Linux, do ktĂłrego przygotowania wykorzystano Änarzdzie Buildroot. Na Ćkocowe Ä
rozwizanie ponadto Ćskada Äsi Ćpoatane Ä
jdro Linuxa, bootloader uboot oraz programowalna logika Ä
realizujca przechwytywanie pakietĂłw sieciowych. Ä
Rozwizanie poddane jest testom, w ktĂłrych parametry odpowiedzialne za ĆĆÄdugo transakcji typu burst, rozmiaru pakietu oraz ÄĆczstotliwoci zegara Ä
s poddawane modyfikacjom. Wyniki Ä
s przedstawione za Ä
pomoc czasu przetwarzania pakietu, czasu per pakiet oraz ĆŒzuycia mocy. Do oceny Ćefektywnoci Ä
rozwizania ĆĆŒĆposuyo ĆŒtake porĂłwnanie z czasem procesowania pakietu w niezmodyfikowanym systemie Linux Na podstawie eksperymentĂłw dokonanych w pracy Äwysunite Ä
s ÄÄ
nastpujce wnioski: wraz ze wzrostem ÄĆczstotliwoci zegara oraz ĆĆdugoci transakcji burst, czas procesowania pakietĂłw maleje a ĆŒzuycie Ä
prdu pozostaje na dotychczasowym poziomie. Pakiety o rozmiarze Ä
przekraczajcym 64 bajty Ä
s procesowane wydajniej w dostarczonym Ä
rozwizaniu poprzez Äkompensacj dodatkowego Ćnakadu czasu narzuconego przez Älogik Ä
Ä
Ä
zarzdzajc przetwarzaniem. System porĂłwnano ĆŒtake do Ćzwykego przetwarzania pakietĂłw Ä
odbywajcego Äsi w systemie Linux ktĂłre Ćokazao Äsi zdecydowanie wolniejsze z ĆŒzastrzeeniem, ĆŒi Ăłw system dokonuje Ćpenego przetworzenia pakietĂłw a Ä
rozwizanie w pracy jedynie ich przechwytywania. Projekt stanowi Äpodstaw do ewentualnych Ćrozszerze, na Ćprzykad filtrowania pakietĂłw. Wnioski Äwysunite ĆĆŒÄ
su ĆÄpogbieniu wiedzy w domenie sieci wbudowanych systemĂłw Linux oraz Äsprztowej akceleracji
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