Functional ramifications for the loss of P-selectin expression on hematopoietic and leukemic stem cells

Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between blood cells and their microenvironment to preserve the homeostatic balance of long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors (MPPs), and differentiated cells. Adhesion molecules like P-selectin (encoded by the Selp gene) are essential to hematopoiesis, and their dysregulation has been linked to leukemogenesis. Like HSCs, leukemic stem cells (LSCs) depend upon their microenvironments for survival and propagation. P-selectin plays a crucial role in Philadelphia chromosome-positive (Ph(+)) chronic myeloid leukemia (CML). In this paper, we show that cells deficient in P-selectin expression can repopulate the marrow more efficiently than wild type controls. This results from an increase in HSC self-renewal rather than alternative possibilities like increased homing velocity or cell cycle defects. We also show that P-selectin expression on LT-HSCs, but not ST-HSCs and MPPs, increases with aging. In the absence of P-selectin expression, mice at 6 months of age possess increased levels of short-term HSCs and multipotent progenitors. By 11 months of age, there is a shift towards increased levels of long-term HSCs. Recipients of BCR-ABL-transduced bone marrow cells from P-selectin-deficient donors develop a more aggressive CML, with increased percentages of LSCs and progenitors. Taken together, our data reveal that P-selectin expression on HSCs and LSCs has important functional ramifications for both hematopoiesis and leukemogenesis, which is most likely attributable to an intrinsic effect on stem cell self-renewal.     

Host cell egress and invasion induce marked relocations of glycolytic enzymes in Toxoplasma gondii tachyzoites

Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K(+)] experienced during egress and invasion, a signal that requires changes of [Ca(2+)](c) in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites.     

Proteolytic processing of TgIMC1 during maturation of the membrane skeleton of Toxoplasma gondii

Membrane skeletons play an important role in the maintenance of cell shape and integrity in many cell types. In the protozoan parasite Toxoplasma gondii this function is performed by the subpellicular network, a resilient structure composed of tightly interwoven 10-nm filaments. We report here that this network is assembled at an early stage in the development of daughter parasites. The networks of immature and mature parasites differ dramatically with respect to their stability. Although in immature parasites the network is completely solubilized by detergent, the network in mature parasites is entirely detergent-resistant. Conversion of the detergent-labile to the detergent-resistant network occurs late in daughter cell development and appears to be coupled to proteolytic processing of the carboxyl terminus of TgIMC1, the major subunit of the network filaments. A single cysteine residue in the TgIMC1 carboxyl terminus was found to be essential for this processing event. The dramatic change in resistance to detergent extraction probably reflects an overall change in structural stability of the subpellicular network that accompanies maturation of daughter parasites and allows a switch from an assembly-competent but loose structure to one that is rigid and offers mechanical strength to the mature parasite.     

A study of AroP-PheP chimeric proteins and identification of a residue involved in tryptophan transport

In vivo recombination has been used to make a series of AroP-PheP chimeric proteins. Analysis of their respective substrate profiles and activities has identified a small region within span III of AroP which can confer on a predominantly PheP protein the ability to transport tryptophan. Site-directed mutagenesis of the AroP-PheP chimera, PheP, and AroP has established that a key residue involved in tryptophan transport is tyrosine at position 103 in AroP. Phenylalanine is the residue at the corresponding position in PheP. The use of PheP-specific antisera has shown that the inability of certain chimeras to transport any of the aromatic amino acids is not a result of instability or a failure to be inserted into the membrane. Site-directed mutagenesis has identified two significant AroP-specific residues, alanine 107 and valine 114, which are the direct cause of loss of transport activity in chimeras such as A152P. These residues replace a glycine and an alanine in PheP and flank a highly conserved glutamate at position 110. Some suggestions are made as to the possible functions of these residues in the tertiary structure of the proteins.     

CSL311, a novel,potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common β chain of the IL-3, GM-CSF and IL-5 receptors

The β common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared β common (β_c, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human β_c receptor. The binding epitope of CSL311 on the β_c receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human β_c receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 β common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human β_c receptor is central to pathogenesis. The coordinates for the β_c/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).     

Ultrastructure of lymphocytes from patients with paracoccidioidomycosis in the lymphocyte transformation test by phytohemagglutinin

The morphology and ultrastructure of peripheral blood lymphocytes from patients with paracoccidioidomycosis (PCM) and from unaffected individuals (controls) were studied before and after Ficoll-Hypaque separation and at the end of culture, stimulated with phytohemagglutinin. Patient lymphocytes were cultured in medium with autologous plasma (from the patient himself) and with homologous plasma (from an unaffected donor), while donor lymphocytes were cultured in medium with plasma from a patient or with plasma from the donor himself. The Ficoll-Hypaque mixture caused no morphological or ultrastructural changes in the lymphocytes of patients or of unaffected donors. Patient lymphocytes cultured in medium with autologous plasma showed different degrees of cytoplasmic and nuclear alterations, such as organelle dissolution, vacuoles, amorphous masses, deformed nuclei, and absence of nucleoli. Lymphocytes from control individuals cultured in patient plasma also showed ultrastructural alterations, though they were less marked, and a reduced number of blasts. Patient lymphocytes cultured in medium with homologous plasma (from a control individual) showed a morphology similar to that of lymphocytes from control individuals cultured in medium with their own plasma, although with a lower number of blasts. On the basis of the results obtained using that methodology, we draw the following conclusions: (1) separation by Ficoll-Hypaque does not seem to alter the ultrastructure of patient or donor lymphocytes; (2) patients with diffuse PCM and more markedly impaired general condition can exhibit lymphocytes with morphological and ultrastructural alterations capable of affecting their biological systems and functionality; (3) the morphological and ultrastructural abnormalities and the reduced blastogenesis observed in patient lymphocytes cultured in autologous plasma and in control lymphocytes cultured with patient plasma appear to be due to factor(s) present in the plasma of PCM patients.     

The use of donor scleral patch in ophthalmic surgery

Scleral tissue has been in use in ophthalmology for many years although indications for use have varied. We retrospectively reviewed scleral transplant tissue requests over a 12 month period at a local eye bank and confirmed a small but significant demand for the use of scleral tissue. Iatrogenic surgical complications are the primary indication for use. Our understanding of the indications and outcomes of scleral graft procedures is derived from case reports and small cohort series. We reviewed the current literature on existing indications for its use and discuss the relative outcomes. To our knowledge this represents the first review of scleral transplant indications and further summarises usage rates in the Lions NSW Eye Bank which may provide practical information for those surgeons who use scleral tissue and Eye Banks who supply it.     

Variations of DNA methylation in Eucalyptus urophylla×Eucalyptus grandis shoot tips and apical meristems of different physiological ages

Global DNA methylation was assessed by high-performance liquid chromatography (HPLC) for the first time in Eucalyptus urophylla×Eucalyptus grandis shoot tips comparing three outdoor and one in vitro sources of related genotypes differing in their physiological age. The DNA methylation levels found were consistent with those reported for other Angiosperms using the same HPLC technology. Notwithstanding noticeable time-related fluctuations within each source of plant material, methylation rate was overall higher for the mature clone (13.7%) than for the rejuvenated line of the same clone (12.6%) and for the juvenile offspring seedlings (11.8%). The in vitro microshoots of the mature clone were less methylated (11.3%) than the other outdoor origins, but the difference with the juvenile seedlings was not significant. Immunofluorescence investigations on shoot apices established that the mature source could be distinguished from the rejuvenated and juvenile origins by a higher density of cells with methylated nuclei in leaf primordia. Shoot apical meristems (SAMs) from the mature clone also showed a greater proportion and more methylated cells than SAMs from the rejuvenated and juvenile origins. The nuclei of these latter were characterized by fewer and more dispersed labeled spots than for the mature source. Our findings establish that physiological ageing induced quantitative and qualitative variations of DNA methylation at shoot tip, SAM and even cellular levels. Overall this DNA methylation increased with maturation and conversely decreased with rejuvenation to reach the lower scores and to show the immunolabeling patterns that characterized juvenile material nuclei.