Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.

Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM-1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins.

Adherence to the first-AUG rule when a second AUG codon follows closely upon the first.

The rule that eukaryotic ribosomes initiate translation exclusively at the 5' proximal AUG codon is abrogated under rare conditions. One circumstance that has been suggested to allow dual initiation is close apposition of a second AUG codon. A possible mechanism might be that the scanning 40S ribosomal subunit flutters back and forth instead of stopping cleanly at the first AUG. This hypothesis seems to be ruled out by evidence presented herein that in certain mRNAs, the first of two close AUG codons is recognized uniquely. To achieve this, the 5' proximal AUG has to be provided with the full consensus sequence; even small departures allow a second nearby AUG codon to be reached by leaky scanning. This context-dependent leaky scanning unexpectedly fails when the second AUG codon is moved some distance from the first. A likely explanation, based on analyzing the accessibility of a far-downstream AUG codon under conditions of initiation versus elongation, is that 80S elongating ribosomes advancing from the 5' proximal start site can mask potential downstream start sites.

MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with a t(11;16)(q23;p13.3)

The recurring translocation t(11;16)(q23;p13.3) has been documented only in cases of acute leukemia or myelodysplasia secondary to therapy with drugs targeting DNA topoisomerase II. We show that the MLL gene is fused to the gene that codes for CBP (CREB-binding protein), the protein that binds specifically to the DNA-binding protein CREB (cAMP response element-binding protein) in this translocation. MLL is fused in-frame to a different exon of CBP in two patients producing chimeric proteins containing the AT-hooks, methyltransferase homology domain, and transcriptional repression domain of MLL fused to the CREB binding domain or to the bromodomain of CBP. Both fusion products retain the histone acetyltransferase domain of CBP and may lead to leukemia by promoting histone acetylation of genomic regions targeted by the MLL AT-hooks, leading to transcriptional deregulation via aberrant chromatin organization. CBP is the first partner gene of MLL containing well defined structural and functional motifs that provide unique insights into the potential mechanisms by which these translocations contribute to leukemogenesis.

Lipoxin A4 stable analogs inhibit leukocyte rolling and adherence in the rat mesenteric microvasculature: Role of P-selectin

Three different stable lipoxin A4 (LXA4) analogs (i.e., 16-phenoxy-LXA4-Me, 15-cyclohexyl-LXA4-Me, and 15-R/S-methyl-LXA4-Me) were studied for their ability to modulate leukocyte-endothelial cell interactions in the rat mesenteric microvasculature. Superfusion of the rat mesentery with 50 μmol/liter NG-nitro-l-arginine methyl ester (l-NAME) caused a significant, time-dependent increase in leukocyte rolling (56 ± 8 cells/min; P < 0.01 vs. control) and leukocyte adherence (12.5 ± 1.2 cells/100 μm length of venule; P < 0.01 vs. control) after 120 min of superfusion. Concomitant superfusion of the rat mesentery with 10 nmol/liter of each of three lipoxin analogs consistently and markedly attenuated l-NAME-induced leukocyte rolling to 10 ± 4 (P < 0.01), 4 ± 1 (P < 0.01), and 32 ± 7 (P < 0.05) cells/min, and adherence to 4 ± 0.8 (P < 0.01), 1.1 ± 0.4 (P < 0.01), and 7 ± 0.7 (P < 0.05) cells/100 μm length of venule (16-phenoxy-LXA4-Me, 15-cyclohexyl-LXA4-Me, and 15-R/S- methyl-LXA4-Me, respectively). No alterations of systemic blood pressure or mesenteric venular shear rates were observed in any group. Immunohistochemical up-regulation of P-selectin expression on intestinal venular endothelium was significantly increased (P < 0.01) after exposure to l-NAME, and this was significantly attenuated by these lipoxin analogs (P < 0.01). Thus, in vivo superfusion of the rat mesentery with stable lipoxin analogs at 10 nmol/liter reduces l-NAME-induced leukocyte rolling and adherence in the mesenteric rat microvasculature by attenuating P-selectin expression. This anti-inflammatory mechanism may represent a novel and potent regulatory action of lipoxins on the immune system.

The control of hematopoiesis and leukemia: from basic biology to the clinic.

Hematopoiesis gives rise to blood cells of different lineages throughout normal life. Abnormalities in this developmental program lead to blood cell diseases including leukemia. The establishment of a cell culture system for the clonal development of hematopoietic cells made it possible to discover proteins that regulate cell viability, multiplication and differentiation of different hematopoietic cell lineages, and the molecular basis of normal and abnormal blood cell development. These regulators include cytokines now called colony-stimulating factors (CSFs) and interleukins (ILs). There is a network of cytokine interactions, which has positive regulators such as CSFs and ILs and negative regulators such as transforming growth factor beta and tumor necrosis factor (TNF). This multigene cytokine network provides flexibility depending on which part of the network is activated and allows amplification of response to a particular stimulus. Malignancy can be suppressed in certain types of leukemic cells by inducing differentiation with cytokines that regulate normal hematopoiesis or with other compounds that use alternative differentiation pathways. This created the basis for the clinical use of differentiation therapy. The suppression of malignancy by inducing differentiation can bypass genetic abnormalities that give rise to malignancy. Different CSFs and ILs suppress programmed cell death (apoptosis) and induce cell multiplication and differentiation, and these processes of development are separately regulated. The same cytokines suppress apoptosis in normal and leukemic cells, including apoptosis induced by irradiation and cytotoxic cancer chemotherapeutic compounds. An excess of cytokines can increase leukemic cell resistance to cytotoxic therapy. The tumor suppressor gene wild-type p53 induces apoptosis that can also be suppressed by cytokines. The oncogene mutant p53 suppresses apoptosis. Hematopoietic cytokines such as granulocyte CSF are now used clinically to correct defects in hematopoiesis, including repair of chemotherapy-associated suppression of normal hematopoiesis in cancer patients, stimulation of normal granulocyte development in patients with infantile congenital agranulocytosis, and increase of hematopoietic precursors for blood cell transplantation. Treatments that decrease the level of apoptosis-suppressing cytokines and downregulate expression of mutant p53 and other apoptosis suppressing genes in cancer cells could improve cytotoxic cancer therapy. The basic studies on hematopoiesis and leukemia have thus provided new approaches to therapy.

Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages.

The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF-alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170-kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal-lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate.

Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension.

Gene targeting allows precise, predetermined changes to be made in a chosen gene in the mouse genome. To date, targeting has been used most often for generation of animals completely lacking the product of a gene of interest. The resulting "knockout" mice have confirmed some hypotheses, have upset others, but have rarely been uninformative. Models of several human genetic diseases have been produced by targeting--including Gaucher disease, cystic fibrosis, and the fragile X syndrome. These diseases are primarily determined by defects in single genes, and their modes of inheritance are well understood. When the disease under study has a complex etiology with multiple genetic and environmental components, the generation of animal models becomes more difficult but no less valuable. The problems associated with dissecting out the individual genetic factors also increases substantially and the distinction between causation and correlation is often difficult. To prove causation in a complex system requires rigorous adherence to the principle that the experiments must allow detection of the effects of changing only a single variable at one time. Gene targeting experiments, when properly designed, can test the effects of a precise genetic change completely free from the effects of differences in any other genes (linked or unlinked to the test gene). They therefore allow proofs of causation.

Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.

Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.