Emerging metabolic targets in the therapy of hematological malignancies

During the last decade, the development of anticancer therapies has focused on targeting neoplastic-related metabolism. Cancer cells display a variety of changes in their metabolism, which enable them to satisfy the high bioenergetic and biosynthetic demands for rapid cell division. One of the crucial alterations is referred to as the "Warburg effect", which involves a metabolic shift from oxidative phosphorylation towards the less efficient glycolysis, independent of the presence of oxygen. Although there are many examples of solid tumors having altered metabolism with high rates of glucose uptake and glycolysis, it was only recently reported that this phenomenon occurs in hematological malignancies. This review presents evidence that targeting the glycolytic pathway at different levels in hematological malignancies can inhibit cancer cell proliferation by restoring normal metabolic conditions. However, to achieve cancer regression, high concentrations of glycolytic inhibitors are used due to limited solubility and biodistribution, which may result in toxicity. Besides using these inhibitors as monotherapies, combinatorial approaches using standard chemotherapeutic agents could display enhanced efficacy at eradicating malignant cells. The identification of the metabolic enzymes critical for hematological cancer cell proliferation and survival appears to be an interesting new approach for the targeted therapy of hematological malignancies.

Dissection of floral pollination syndromes in petunia

Animal-mediated pollination is essential in the reproductive biology of many flowering plants and tends to be associated with pollination syndromes, sets of floral traits that are adapted to particular groups of pollinators. The complexity and functional convergence of various traits within pollination syndromes are outstanding examples of biological adaptation, raising questions about their mechanisms and origins. In the genus Petunia, complex pollination syndromes are found for nocturnal hawkmoths (P. axillaris) and diurnal bees (P. integrifolia), with characteristic differences in petal color, corolla shape, reproductive organ morphology, nectar quantity, nectar quality, and fragrance. We dissected the Petunia syndromes into their most important phenotypic and genetic components. They appear to include several distinct differences, such as cell-growth and cell-division patterns in the basal third of the petals, elongation of the ventral stamens, nectar secretion and nectar sugar metabolism, and enzymatic differentiation in the phenylpropanoid pathway. In backcross-inbred lines of species-derived chromosome segments in a transposon tagging strain of P. hybrida, one to five quantitative trait loci were identified for each syndrome component. Two loci for stamen elongation and nectar volume were confirmed in introgression lines and showed large allelic differences. The combined data provide a framework for a detailed understanding of floral syndromes from their developmental and molecular basis to their impact on animal behavior. With its molecular genetic tools, this Petunia system provides a novel venue for a pattern of adaptive radiation that is among the most characteristic of flowering plants.

The orf13 T-DNA gene of Agrobacterium rhizogenes confers meristematic competence to differentiated cells

Plant infections by the soil bacterium Agrobacterium rhizogenes result in neoplastic disease with the formation of hairy roots at the site of infection. Expression of a set of oncogenes residing on the stably integrated T-DNA is responsible for the disease symptoms. Besides the rol (root locus) genes, which are essential for the formation of hairy roots, the open reading frame orf13 mediates cytokinin-like effects, suggesting an interaction with hormone signaling pathways. Here we show that ORF13 induced ectopic expression of KNOX (KNOTTED1-like homeobox) class transcription factors, as well as of several genes involved in cell cycle control in tomato (Lycopersicon esculentum). ORF13 has a retinoblastoma (RB)-binding motif and interacted with maize (Zea mays) RB in vitro, whereas ORF13, bearing a point mutation in the RB-binding motif (ORF13*), did not. Increased cell divisions in the vegetative shoot apical meristem and accelerated formation of leaf primordia were observed in plants expressing orf13, whereas the expression of orf13* had no influence on cell division rates in the shoot apical meristem, suggesting a role of RB in the regulation of the cell cycle in meristematic tissues. On the other hand, ectopic expression of LeT6 was not dependent on a functional RB-binding motif. Hormone homeostasis was only altered in explants of leaves, whereas in the root no effects were observed. We suggest that ORF13 confers meristematic competence to cells infected by A. rhizogenes by inducing the expression of KNOX genes and promotes the transition of infected cells from the G1 to the S phase by binding to RB.

Domains of expansin gene expression define growth regions in the shoot apex of tomato

Expansins are members of a multigene family of extracellular proteins, which increase cell wall extensibility in vitro and thus are thought to be involved in cell expansion. The major significance of the presence of this large gene family may be that distinctly expressed genes can independently regulate cell expansion in place and time. Here we report on LeExp9, a new expansin gene from tomato, and compare its expression in the shoot tip with that of LeExp2 and LeExp18. LeExp18 gene is expressed in very young tissues of the tomato shoot apex and the transcript levels are upregulated in the incipient primordium. LeExp2 mRNA accumulated in more mature tissues and transcript levels correlated with cell elongation in the elongation zone. In situ hybridization experiments showed a uniform distribution of LeExp9 mRNA in submeristematic tissues. When gibberellin-deficient mutant tomatoes that lacked elongation of the internodes were treated with gibberellin, the phenotypic rescue was correlated with an increase in LeExp9 and LeExp2, but not LeExp18 levels. We propose that the three expansins define three distinct growing zones in the shoot tip. In the meristem proper, gibberellin-independent LeExp18 mediates the cell expansion that accompanies cell division. In the submeristematic zone, LeExp9 mediates cell expansion at a time that cell division comes to a halt. LeExp9 expression requires gibberellin but the hormone is not normally limiting. Finally, LeExp2 mediates cell elongation in young stem tissue. LeExp2 expression is limited by the available gibberellin. These data suggest that regulation of cell wall extensibility is controlled, at least in part, by differential regulation of expansin genes.

Plant architecture

Plant architecture is species specific, indicating that it is under strict genetic control. Although it is also influenced by environmental conditions such as light, temperature, humidity and nutrient status, here we wish to focus only on the endogenous regulatory principles that control plant architecture. We summarise recent progress in the understanding of the basic patterning mechanisms involved in the regulation of leaf arrangement, the genetic regulation of meristem determinacy, i.e. the decision to stop or continue growth, and the control of branching during vegetative and generative development. Finally, we discuss the basis of leaf architecture and the role of cell division and cell growth in morphogenesis.

Engraftment of autologous bone marrow cells into the injured cranial cruciate ligament in dogs

Current research indicates that exogenous stem cells may accelerate reparative processes in joint disease but, no previous studies have evaluated whether bone marrow cells (BMCs) target the injured cranial cruciate ligament (CCL) in dogs. The objective of this study was to investigate engraftment of BMCs following intra-articular injection in dogs with spontaneous CCL injury. Autologous PKH26-labelled BMCs were injected into the stifle joint of eight client-owned dogs with CCL rupture. The effects of PKH26 staining on cell viability and PKH26 fluorescence intensity were analysed in vitro using a MTT assay and flow cytometry. Labelled BMCs in injured CCL tissue were identified using fluorescence microscopy of biopsies harvested 3 and 13 days after intra-articular BMC injection. The intensity of PKH26 fluorescence declines with cell division but was still detectable after 16 days. Labelling with PKH26 had no detectable effect on cell viability or proliferation. Only rare PKH26-positive cells were present in biopsies of the injured CCL in 3/7 dogs and in synovial fluid in 1/7 dogs. No differences in transforming growth factor-beta1, and interleukin-6 before and after BMC treatment were found and no clinical complications were noted during a 1 year follow-up period. In conclusion, BMCs were shown to engraft to the injured CCL in dogs when injected into the articular cavity. Intra-articular application of PKH26-labelled cultured mesenchymal stem cells is likely to result in higher numbers of engrafted cells that can be tracked using this method in a clinical setting.

Functional proteomics and biochemsitry: working together to unravel mitochondrial phenomena of African trypansomes

Eukaryotic cells are compartmentalized into membrane-bound organelles in order to provide sheltered reaction rooms for various specific processes. Organelles are not randomly distributed in a cell or operate isolated from each other. At the contrary — some organelles are closely linked and their functions are tightly orchestrated. The most well-known example of two such organelles acting in concert are the ER and the mitochondrion that work together in order to coordinate cellular lipid biosynthesis, maintain Ca2+-homeostasis, regulate mitochondrial division and control mitochondrial/ER shape as well as to synchronize the movement of these organelles within a cell. To study the mitochondrion and its interface to the ER requires a simplified mitochondrial system. African trypanosomes represent such a system. The unicellular parasite that causes devastating diseases in humans and animals has only one large mitochondrion that does not undergo fission/fusion events except for the context of cell division. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the protozoan. Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal MOM proteome consists of 82 proteins, two thirds of which have never been associated with mitochondria before. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. Interestingly, a putative tethering complex in mammalian cells was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating, if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved. This ER-subdomain associated with mitochondria has been termed mitochondria-associated ER-membranes or MAM. The MAM has recently been implicated to play a key role in Alzheimer’s disease. It is therefore of broad and general interest to establish other eukaryotic model systems in order to investigate the MAM-MOM connection in more detail.

Nuclear Factor I-C acts as a regulator of hepatocyte proliferation at the onset of liver regeneration

BACKGROUND & AIMS: Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed abnormal skin wound healing and growth of its appendages, suggesting a role in controlling cell proliferation in adult regenerative processes. Liver regeneration following partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes lead to their rapid and phased proliferation. Although NFI-C is highly expressed in the liver, no hepatic function was yet established for this transcription factor. This study aimed to determine whether NFI-C may play a role in hepatocyte proliferation and liver regeneration. METHODS: Liver regeneration and cell proliferation pathways following two-thirds PH were investigated in NFI-C knockout (ko) and wild-type (wt) mice. RESULTS: We show that the absence of NFI-C impaired hepatocyte proliferation because of plasminogen activator I (PAI-1) overexpression and the subsequent suppression of urokinase plasminogen activator (uPA) activity and hepatocyte growth factor (HGF) signalling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wt mice. The subsequent transient down regulation of NFI-C, as can be explained by a self-regulatory feedback loop with transforming growth factor beta 1 (TGF-ß1), may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. CONCLUSION: NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

Functional proteomics and biochemistry: working together to unravel mitochondrial phenomena of African trypanosomes

Eukaryotic cells are compartmentalized into membrane-bound organelles in order to provide sheltered reaction rooms for various specific processes. Organelles are not randomly distributed in a cell or operate isolated from each other. At the contrary — some organelles are closely linked and their functions are tightly orchestrated. The most well-known example of two such organelles acting in concert are the ER and the mitochondrion that work together in order to coordinate cellular lipid biosynthesis, maintain Ca2+-homeostasis, regulate mitochondrial division and control mitochondrial/ER shape as well as to synchronize the movement of these organelles within a cell. To study the mitochondrion and its interface to the ER requires a simplified mitochondrial system. African trypanosomes represent such a system. The unicellular parasite that causes devastating diseases in humans and animals has only one large mitochondrion that does not undergo fission/fusion events except for the context of cell division. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the protozoan. Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal MOM proteome consists of 82 proteins, two thirds of which have never been associated with mitochondria before. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. Interestingly, a putative tethering complex in mammalian cells was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating, if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved. This ER-subdomain associated with mitochondria has been termed mitochondria-associated ER-membranes or MAM. The MAM has recently been implicated to play a key role in Alzheimer’s disease. It is therefore of broad and general interest to establish other eukaryotic model systems in order to investigate the MAM-MOM connection in more detail.

TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization.

Alteration of host cell phenotype by Theileria annulata and Theileria parva: mining for manipulators in the parasite genomes

The apicomplexan parasites Theileria annulata and Theileria parva cause severe lymphoproliferative disorders in cattle. Disease pathogenesis is linked to the ability of the parasite to transform the infected host cell (leukocyte) and induce uncontrolled proliferation. It is known that transformation involves parasite dependent perturbation of leukocyte signal transduction pathways that regulate apoptosis, division and gene expression, and there is evidence for the translocation of Theileria DNA binding proteins to the host cell nucleus. However, the parasite factors responsible for the inhibition of host cell apoptosis, or induction of host cell proliferation are unknown. The recent derivation of the complete genome sequence for both T. annulata and T. parva has provided a wealth of information that can be searched to identify molecules with the potential to subvert host cell regulatory pathways. This review summarizes current knowledge of the mechanisms used by Theileria parasites to transform the host cell, and highlights recent work that has mined the Theileria genomes to identify candidate manipulators of host cell phenotype.

ADAMTS13 activity in sickle cell disease

Sickle red blood cell (SRBC)-endothelial adhesion plays a central role in sickle cell disease (SCD)-related vaso-occlusion. As unusually large von Willebrand factor (ULVWF) multimers mediate SRBC-endothelial adhesion, we investigated the activity of ADAMTS13, the metalloprotease responsible for cleaving ULVWF multimers, in SCD. ADAMTS13 activity was determined using a quantitative immunoblotting assay. VWF:Ag and VWF:RCo were determined using commercial assays. The high-molecular-weight VWF multimer percentage was determined by employing gel electrophoresis. ADAMTS13 activity was similar among asymptomatic patients (n = 8), patients at presentation with a painful crisis (n = 23), and healthy controls. ADAMTS13/VWF:Ag ratios were lower in patients compared to healthy HbAA controls, with the lowest values at presentation with a painful crisis (P = 0.02). Division of samples in those with VWF:RCo/VWF:Ag ratios < 0.70 and those with ratios >or= 0.70 revealed significantly more samples with ratios >or= 0.70 (P = 0.01) collected during painful crises. ULVWF multimers were detected in 6 patient samples and in 1 control sample. ADAMTS13/VWF:Ag ratios were inversely related to the duration of symptoms at presentation with an acute vaso-occlusive event (r(s)-0.67, P = 0.002). Although SCD is characterized by elevated VWF:Ag levels, no severe ADAMTS13 deficiency was detected in our patients.

A Drosophila XPD model links cell cycle coordination with neuro-development and suggests links to cancer

XPD functions in transcription, DNA repair and in cell cycle control. Mutations in human XPD (also known as ERCC2) mainly cause three clinical phenotypes: xeroderma pigmentosum (XP), Cockayne syndrome (XP/CS) and trichothiodystrophy (TTD), and only XP patients have a high predisposition to developing cancer. Hence, we developed a fly model to obtain novel insights into the defects caused by individual hypomorphic alleles identified in human XP-D patients. This model revealed that the mutations that displayed the greatest in vivo UV sensitivity in Drosophila did not correlate with those that led to tumor formation in humans. Immunoprecipitations followed by targeted quantitative MS/MS analysis showed how different xpd mutations affected the formation or stability of different transcription factor IIH (TFIIH) subcomplexes. The XP mutants most clearly linked to high cancer risk, Xpd R683W and R601L, showed a reduced interaction with the core TFIIH and also an abnormal interaction with the Cdk-activating kinase (CAK) complex. Interestingly, these two XP alleles additionally displayed high levels of chromatin loss and free centrosomes during the rapid nuclear division phase of the Drosophila embryo. Finally, the xpd mutations showing defects in the coordination of cell cycle timing during the Drosophila embryonic divisions correlated with those human mutations that cause the neurodevelopmental abnormalities and developmental growth defects observed in XP/CS and TTD patients.