Prognostic relevance of dic(9;20)(p11;q13) in childhood B‐cell precursor acute lymphoblastic leukaemia treated with Berlin‐Frankfurt‐Münster (BFM) protocols containing an intensive induction and post‐induction consolidation therapy

The presence of a dicentric chromosome dic(9;20) has been reported to have an unfavourable prognosis in children with B‐cell precursor acute lymphoblastic leukaemia (BCP‐ALL). As outcome may be influenced by type and composition of treatment, we analyzed 19 BCP‐ALL patients with dic(9;20) who have been treated with ALL‐BFM (Berlin‐Frankfurt‐Münster) protocols that included a 4‐drug induction and subsequent consolidation therapy. All patients were good responders to prednisone and in complete remission after induction therapy. Eight patients had no molecular disease after induction and another eight patients had levels ≤10^?4 after consolidation therapy. After a median follow‐up of 3·4 years, probabilities of 5‐year event‐free and overall survival were 75 ± 11% and 94 ± 6%, respectively. Of note, there was a tendency for extramedullary disease in case of relapse (two of three relapses with central nervous system involvement). In conclusion, in the context of ALL‐BFM protocols dic(9;20)‐positivity appeared to have a favourable prognosis, which could be due to a dose‐ and time‐intensified induction and induction consolidation therapy. Given that in vitro studies have shown high cellular sensitivity of dic(9;20)‐positive leukemic blasts to l ‐asparaginase and cytarabine, it is reasonable to speculate that both drugs, as given early during BFM‐like induction and consolidation therapy, may have contributed to this good outcome.     

Generation of trisomies in cancer cells by multipolar mitosis and incomplete cytokinesis

One extra chromosome copy (i.e., trisomy) is the most common type of chromosome aberration in cancer cells. The mechanisms behind the generation of trisomies in tumor cells are largely unknown, although it has been suggested that dysfunction of the spindle assembly checkpoint (SAC) leads to an accumulation of trisomies through failure to correctly segregate sister chromatids in successive cell divisions. By using Wilms tumor as a model for cancers with trisomies, we now show that trisomic cells can form even in the presence of a functional SAC through tripolar cell divisions in which sister chromatid separation proceeds in a regular fashion, but cytokinesis failure nevertheless leads to an asymmetrical segregation of chromosomes into two daughter cells. A model for the generation of trisomies by such asymmetrical cell division accurately predicted several features of clones having extra chromosomes in vivo, including the ratio between trisomies and tetrasomies and the observation that different trisomies found in the same tumor occupy identical proportions of cells and colocalize in tumor tissue. Our findings provide an experimentally validated model explaining how multiple trisomies can occur in tumor cells that still maintain accurate sister chromatid separation at metaphase-anaphase transition and thereby physiologically satisfy the SAC.     

The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis

The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana, called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core proteins. PPH1, which belongs to the family of monomeric PP2C type phosphatases, is a chloroplast protein and is mainly associated with the stroma lamellae of the thylakoid membranes. We demonstrate that loss of PPH1 leads to an increase in the antenna size of photosystem I and to a strong impairment of state transitions. Thus phosphorylation and dephosphorylation of LHCII appear to be specifically mediated by the kinase/phosphatase pair STN7 and PPH1. These two proteins emerge as key players in the adaptation of the photosynthetic apparatus to changes in light quality and quantity.     

Induction of cortical endoplasmic reticulum by dimerization of a coatomer-binding peptide anchored to endoplasmic reticulum membranes

Cortical endoplasmic reticulum (cER) is a permanent feature of yeast cells but occurs transiently in most animal cell types. Ist2p is a transmembrane protein that permanently localizes to the cER in yeast. When Ist2 is expressed in mammalian cells, it induces abundant cER containing Ist2. Ist2 cytoplasmic C-terminal peptide is necessary and sufficient to induce cER. This peptide sequence resembles classic coat protein complex I (COPI) coatomer protein-binding KKXX signals, and indeed the dimerized peptide binds COPI in vitro. Controlled dimerization of this peptide induces cER in cells. RNA interference experiments confirm that coatomer is required for cER induction in vivo, as are microtubules and the microtubule plus-end binding protein EB1. We suggest that Ist2 dimerization triggers coatomer binding and clustering of this protein into domains that traffic at the microtubule growing plus-end to generate the cER beneath the plasma membrane. Sequences similar to the Ist2 lysine-rich tail are found in mammalian STIM proteins that reversibly induce the formation of cER under calcium control.The current view of the yeast endoplasmic reticulum (ER) discriminates perinuclear ER from cortical ER (cER), which forms a circular structure apposed to the plasma membrane (PM) (1). Both structures are connected by tubulated membranes (2, 3), at least transiently, because ER membranes undergo continuous fission and fusion events (4, 5). cER is a much less prominent feature of most mammalian cells (6). The best-characterized function of cER is its role in the store-operated calcium entry, an ubiquitous Ca²⁺ influx pathway activated in response to depletion of intracellular calcium stores (7).Ist2 is a “yeast peripheral” protein involved in osmotic stress tolerance. It was initially believed to be located at the plasma membrane (8–11), and its cytosolic tail (Ist2ct) has been shown to carry the peripheral targeting signal (8). Ist2ct includes a dimerization domain (amino acids 878–928) and a lysine-rich carboxy terminal tail containing a KKXX-like motif that has been proposed to interact with the PM (11, 12). The nature of the peripheral Ist2 resident sites remains a matter of debate, however. It was once thought that Ist2 reached the PM in a new Golgi-independent manner (10), but more recently it has been concluded that the major residence site is in fact the cER (11).To gain insight into the biogenesis of cER in mammalian cells, we investigated whether Ist2, when expressed in a heterologous system, can serve as a useful marker for this compartment. Interestingly, enrichment of Ist2 chimeric protein at the cER appears to directly modulate the formation and/or maintenance of this ER subdomain. These dynamic changes in peripheral ER structure are absolutely dependent on both microtubules and coat protein complex I (COPI) and suggest a different role of COPI than its classical one.     

Endoplasmic reticulum chaperone gp96 is essential for infection with vesicular stomatitis virus

The envelope glycoprotein of vesicular stomatitis virus (VSV-G) enables viral entry into hosts as distant as insects and vertebrates. Because of its ability to support infection of most, if not all, human cell types VSV-G is used in viral vectors for gene therapy. However, neither the receptor nor any specific host factor for VSV-G has been identified. Here we demonstrate that infection with VSV and innate immunity via Toll-like receptors (TLRs) require a shared component, the endoplasmic reticulum chaperone gp96. Cells without gp96 or with catalytically inactive gp96 do not bind VSV-G. The ubiquitous expression of gp96 is therefore essential for the remarkably broad tropism of VSV-G. Cells deficient in gp96 also lack functional TLRs, which suggests that pathogen-driven pressure for TLR-mediated immunity maintains the broad host range of VSV-G by positively selecting for the ubiquitous expression of gp96.     

Distant Relatives of Severe Acute Respiratory Syndrome Coronavirus and Close Relatives of Human Coronavirus 229E in Bats, Ghana

We tested 12 bat species in Ghana for coronavirus (CoV) RNA. The virus prevalence in insectivorous bats (n = 123) was 9.76%. CoV was not detected in 212 fecal samples from Eidolon helvum fruit bats. Leaf-nosed bats pertaining to Hipposideros ruber by morphology had group 1 and group 2 CoVs. Virus concentrations were <45,000 copies/100 mg of bat feces. The diversified group 1 CoV shared a common ancestor with the human common cold virus hCoV-229E but not with hCoV-NL63, disputing hypotheses of common human descent. The most recent common ancestor of hCoV-229E and GhanaBt-CoVGrp1 existed in ≈1686–1800 ad. The GhanaBt-CoVGrp2 shared an old ancestor (≈2,400 years) with the severe acute respiratory syndrome–like group of CoV.     

Inhibitory activity of quercetin and its metabolite on lipopolysaccharide-induced activation of macrophage U937 cells

The potential of quercetin and its metabolite 3-O-methyl quercetin in inhibiting lipopolysaccharide (LPS)-mediated activation of macrophage U937 cells was investigated. Cells were pre-incubated for different periods with 100 ng/mL phorbol myristate acetate (PMA), and later with LPS and quercetin or 3-O-methyl quercetin (30 μM). Later, the supernatant of each cell culture was assessed for catalase activity, nitric oxide, and the production of tumour necrosis factor-α (TNF-α), interleukin 6 (IL-6), and interleukin 1 (IL-1). The results showed that when the cells were incubated with LPS, there were elevations in the levels of all the markers over the cells not incubated with LPS (P < 0.05). For the cells that were incubated with LPS, there were significant differences between the various cells when they were pre-incubated with PMA for various periods (P < 0.05). However, greatest production of the markers was attained when the cells were pre-treated with PMA for 48 h. Both quercetin and 3-O-methyl quercetin (at 30 mM) reduced the levels of all the markers with 3-O-methyl quercetin possessing more inhibitory potential (P < 0.05). This suggests that the flavonoids possessed significant immunomodulatory activities which depend on methylation especially at position 3.     

Oral immunization of raccoons and skunks with a canine adenovirus recombinant rabies vaccine

Oral vaccination is an important part of wildlife rabies control programs. Currently, the vaccinia-rabies glycoprotein recombinant virus is the only oral rabies vaccine licensed in the United States, and it is not effective in skunks. In the current study, captive raccoons and skunks were used to evaluate a vaccine developed by incorporating the rabies virus glycoprotein gene into a canine adenovirus serotype 2 vector (CAV2-RVG). Seven of 7 raccoons orally vaccinated with CAV2-RVG developed virus neutralizing antibodies and survived lethal challenge. Five of 5 and 6 of 6 skunks in 2 experimental groups receiving 10-fold different dilutions of CAV2-RVG developed neutralizing antibodies and survived challenge. The results of this preliminary study suggest that CAV2-RVG stimulates protective immunity against rabies in raccoons and skunks.