Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to fas-induced apoptosis

Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.     

Crystal structure and biochemical properties of the D-arabinose dehydrogenase from Sulfolobus solfataricus

Sulfolobus solfataricus metabolizes the five-carbon sugar d-arabinose to 2-oxoglutarate by an inducible pathway consisting of dehydrogenases and dehydratases. Here we report the crystal structure and biochemical properties of the first enzyme of this pathway: the d-arabinose dehydrogenase. The AraDH structure was solved to a resolution of 1.80 A by single-wavelength anomalous diffraction and phased using the two endogenous zinc ions per subunit. The structure revealed a catalytic and cofactor binding domain, typically present in mesophilic and thermophilic alcohol dehydrogenases. Cofactor modeling showed the presence of a phosphate binding pocket sequence motif (SRS-X2-H), which is likely to be responsible for the enzyme's preference for NADP+. The homo-tetrameric enzyme is specific for d-arabinose, l-fucose, l-galactose and d-ribose, which could be explained by the hydrogen bonding patterns of the C3 and C4 hydroxyl groups observed in substrate docking simulations. The enzyme optimally converts sugars at pH 8.2 and 91 degrees C, and displays a half-life of 42 and 26 min at 85 and 90 degrees C, respectively, indicating that the enzyme is thermostable at physiological operating temperatures of 80 degrees C. The structure represents the first crystal structure of an NADP+-dependent member of the medium-chain dehydrogenase/reductase (MDR) superfamily from Archaea.     

Retrotransposon insertion-site context (RISC) typing: a novel typing method for Aspergillus fumigatus and a convenient PCR alternative to restriction fragment length polymorphism analysis

Retrotransposon(-like) sequences in Aspergillus fumigatus have been used as typing targets through restriction fragment length polymorphism (RFLP)/Southern blotting approaches. Differences in fingerprints between unrelated isolates are the result of variations in copy-number and differences in the regions flanking the retrotransposon elements. Here, we present retrotransposon insertion-site context (RISC) typing as a novel and convenient PCR-based typing alternative to the RFLP approach. RISC typing aims at amplifying the sequences flanking the retrotransposon-like sequences in A. fumigatus and allows large numbers of isolates to be analyzed in a timely fashion with excellent discriminatory power.     

Nutritional imbalance caused by nitrogen excess is correlated with the occurrence of watermark disease in white willow

Willows in the Flemish region are threatened by the watermark disease, a wood disease caused by the bacterium Brenneria salicis (Hauben et al. Syst Appl Microbiol 21:384–397, 1998). Affected trees are observed to grow at specific sites which seem more sensitive to periodical watermark symptom development than other sites. In this study we aim at revealing abiotic site factors that could play a role in triggering the watermark disease. During three consecutive years we analyzed stands of young isogenic willows (Salix alba L. cv. Lichtenvoorde), which were planted on four different sites in an agricultural environment. These test sites were chosen, based on the occurrence/absence of watermark disease in the old willows that grow on these sites. Two sites had old willow pollards with clear watermark symptoms, and on the two other sites the old willows were unaffected. Differences in abiotic characteristics between the sites were analysed and their effects on the nutrient status in willow were measured in the isogenic willows that were newly planted. Brenneria salicis-specific multiplex PCR revealed the presence of this pathogenic bacterium in both the old and the freshly planted young willows. At each site, the physical and chemical characteristics of soil and groundwater were investigated; the young tree growth was monitored and nutritional status of the willow plantings was assessed by foliar analysis. Physical soil properties including soil type and profile water availability, and chemical soil analysis were unable to explain differences between healthy and affected sites. Diseased sites, however, had significantly higher N concentrations and traces of herbicides in the groundwater. In young trees, foliar levels of N and S were always greater at the affected sites, while P contents were lower. Dry mass based N concentration in the leaves of the newly planted trees increased systematically over the years, but more at affected than at healthy sites. Also, chlorophyll content was significantly greater at the affected sites. Growth rate of the young isogenic trees was twice as fast at the diseased sites. The presented data suggest that application of fertilisers in agriculture causes an imbalance in the nutritional status of Salix alba L. Excess nitrogen makes this species fast-growing, reflected by an average foliar N:P ratio >10, and more susceptible for watermark disease, indicated by an average foliar N:Ca ratio exceeding 1.5.     

MK3 Modulation Affects BMI1-Dependent and Independent Cell Cycle Check-Points

Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16^INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.     

Upregulation of IGF-1R Expression during Neoadjuvant Therapy Predicts Poor Outcome in Breast Cancer Patients

IntroductionThe insulin-like growth factor 1 receptor (IGF-1R) may be involved in the development of resistance against conventional cancer treatment. The aim of this study was to assess whether IGF-1R expression of breast tumors changes during neoadjuvant therapy and to study whether these changes were associated with survival.MethodsParaffin embedded tumor tissue was collected from pretreatment biopsies and surgical resections of 62 breast cancer patients who were treated with neoadjuvant chemotherapy or endocrine therapy. IGF-1R expression was determined immunohistochemically and compared before and after treatment.ResultsHigh membranous IGF-1R expression at diagnosis correlated significantly with ER positivity, low tumor stage (stage I/II) and longer overall survival (p < 0.05). After neoadjuvant treatment, membranous IGF-1R expression remained the same in 41 (65%) tumors, was upregulated in 11 (18%) tumors and downregulated in 11 (18%) tumors. Changes in membranous IGF-1R expression were associated with overall survival (log-rank test: p = 0.013, multivariate cox-regression: p = 0.086). Mean overall survival time for upregulation, no change, and downregulation in IGF-1R expression was 3.0 ± 0.5 years, 7.3 ± 1.0 years and 15.0 ± 1.8 years, respectively. Changes in other parameters were not significantly associated with survival.ConclusionNeoadjuvant therapy can induce changes in IGF-1R expression. Upregulation of IGF-1R expression after neoadjuvant treatment is a poor prognostic factor in breast cancer patients, providing a rationale for incorporating anti-IGF-1R drugs in the management of these patients.     

Myofascial force transmission also occurs between antagonistic muscles located within opposite compartments of the rat lower hind limb

Force transmission via pathways other than myotendinous ones, is referred to as myofascial force transmission. The present study shows that myofascial force transmission occurs not only between adjacent synergistic muscles or antagonistic muscles in adjacent compartments, but also between most distant antagonistic muscles within a segment. Tibialis anterior (TA), extensor hallucis longus (EHL), extensor digitorum longus (EDL), peroneal muscles (PER) and triceps surae muscles of 7 male anaesthetised Wistar rats were attached to force transducers, while connective tissues at the muscle bellies were left fully intact. The TA + EHL-complex was made to exerted force at different lengths, but the other muscles were held at a constant muscle–tendon complex length. With increasing TA + EHL-complex length, active force of maximally activated EDL, PER and triceps surae decreased by maximally 5%, 32% and 16%, respectively. These decreases are for the largest part explained by myofascial force transmission. Particularly the force decrease in triceps surae muscles is remarkable, because these muscles are located furthest away from the TA + EHL-complex. It is concluded that substantial extramuscular myofascial force transmission occurs between antagonistic muscles even if the length of the path between them is considerable.     

Myofascial force transmission between antagonistic rat lower limb muscles: Effects of single muscle or muscle group lengthening

Effects of lengthening of the whole group of anterior crural muscles (tibialis anterior and extensor hallucis longus muscles (TA + EHL) and extensor digitorum longus (EDL)) on myofascial interaction between synergistic EDL and TA + EHL muscles, and on myofascial force transmission between anterior crural and antagonistic peroneal muscles, were investigated. All muscles were either passive or maximally active. Peroneal muscles were kept at a constant muscle tendon complex length. Either EDL or all anterior crural muscles were lengthened so that effects of lengthening of TA + EHL could be analyzed. For both lengthening conditions, a significant difference in proximally and distally measured EDL passive and active forces, indicative of epimuscular myofascial force transmission, was present. However, added lengthening of TA + EHL significantly affected the magnitude of the active and passive load exerted on EDL. For the active condition, the direction of the epimuscular load on EDL was affected; at all muscle lengths a proximally directed load was exerted on EDL, which decreased at higher muscle lengths. Lengthening of anterior crural muscles caused a 26% decrease in peroneal active force.

Extramuscular myofascial connections are thought to be the major contributor to the EDL proximo-distal active force difference. For antagonistic peroneal complex, the added distal lengthening of a synergistic muscle increases the effects of extramuscular myofascial force transmission.     

Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner

Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase IIIalpha (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is indispensable for ligation of NER-induced breaks and repair of UV lesions in quiescent cells. Furthermore, our results demonstrate that two distinct complexes differentially carry out gap filling in NER. XRCC1-Lig3 and DNA polymerase delta colocalize and interact with NER components in a UV- and incision-dependent manner throughout the cell cycle. In contrast, DNA ligase I and DNA polymerase varepsilon are recruited to UV-damage sites only in proliferating cells. This study reveals an unexpected and key role for XRCC1-Lig3 in maintenance of genomic integrity by NER in both dividing and nondividing cells and provides evidence for cell-cycle regulation of NER-mediated repair synthesis in vivo.     

Delivery of dsRNA for RNAi in insects: an overview and future directions

Abstract RNA interference (RNAi) refers to the process of exogenous double‐stranded RNA (dsRNA) silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi‐based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.