Bone marrow stromal proteoglycans regulate megakaryocytic differentiation of human progenitor cells.

Adherence of hematopoietic progenitor cells (HPCs) to stroma is an important regulatory step in megakaryocytic differentiation. However, the mechanisms through which megakaryocytic progenitors are inhibited by stroma are poorly understood. We examined the role of sulfated glycoconjugates, such as proteoglycans (PGs), on human bone marrow stroma (hBMS).

AtToc90, a new GTP-binding component of the Arabidopsis chloroplast protein import machinery.

AtToc159 is a GTP-binding chloroplast protein import receptor. In vivo, atToc159 is required for massive accumulation of photosynthetic proteins during chloroplast biogenesis. Yet, in mutants lacking atToc159 photosynthetic proteins still accumulate, but at strongly reduced levels whereas non-photosynthetic proteins are imported normally: This suggests a role for the homologues of atToc159 (atToc132, -120 and -90).

Chloroplast protein import: solve the GTPase riddle for entry.

The fidelity of the numerous intracellular protein-trafficking pathways to different organelles is dictated by the interactions between the intrinsic targeting signals of substrate proteins and specific receptors that deliver the substrate to the proper organelle. Recent studies of protein targeting to chloroplasts suggest a novel mechanism in which GTP-dependent substrate recognition is coupled to a GTP-driven motor that initiates the translocation of proteins into the organelle.

Glucuronidation and the UDP-glucuronosyltransferases in health and disease.

This article is an updated report of a symposium held at the June 2000 annual meeting of the American Society for Pharmacology and Experimental Therapeutics in Boston. The symposium was sponsored by the ASPET Divisions for Drug Metabolism and Molecular Pharmacology. The report covers research from the authors' laboratories on the structure and regulation of UDP-glucuronosyltransferase (UGT) genes, glucuronidation of xenobiotics and endobiotics, the toxicological relevance of UGTs, the role of UGT polymorphisms in cancer susceptibility, and gene therapy for UGT deficiencies.

In vitro model for hematopoietic progenitor cell homing reveals endothelial heparan sulfate proteoglycans as direct adhesive ligands.

Proteoglycans (PGs) play a dominant role within the bone marrow (BM), but their role in homing of transplanted hematopoietic progenitor cells (HPC) is unknown. In this study, the role of heparan sulfate (HS) PGs on BM endothelium as adhesive structures was investigated. HPC (primary CD34+ cells and cell line KG-1a) were able to bind fractionated heparin, which could be competed by highly sulfated heparin/HS-glycosaminoglycans (GAGs).

Differential regulation of alternate UDP-glucuronosyltransferase 1A6 gene promoters by hepatic nuclear factor-1.

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major UGT contributing to the glucuronidation of small phenolic compounds. The gene for rat 1A6 is expressed using two promoters, a distal promoter P1 and a proximal promoter P2. Transcripts from P2 are high in liver, gastrointestinal tract, and kidney, whereas P1 transcripts predominate in other tissues.

atTic110 functions as a scaffold for coordinating the stromal events of protein import into chloroplasts.

The translocon of the inner envelope membrane of chloroplasts (Tic) mediates the late events in the translocation of nucleus-encoded preproteins into chloroplasts. Tic110 is a major integral membrane component of active Tic complexes and has been proposed to function as a docking site for translocation-associated stromal factors and as a component of the protein-conducting channel. To investigate the various proposed functions of Tic110, we have investigated the structure, topology, and activities of a 97.

Dimerization of Toc-GTPases at the chloroplast protein import machinery.

Import of chloroplast precursor proteins is controlled by the coordinate action of two homologous GTPases, Toc159 and Toc33, located at the cytosol-outer membrane interface. Recent studies in Arabidopsis showed that the cytosolic form of the precursor binding protein Toc159 is targeted to its receptor at the import machinery, Toc33, via heterodimerization of their GTP-binding domains. Toc33 may also form GDP-bound homodimers, as suggested by the crystal structure of its pea ortholog.

Preliminary estimates of human immunodeficiency virus prevalence and incidence among cocaine abusers of Porto Alegre, Brazil.

A sample of 193 southern Brazilian inner-city cocaine abusers was studied to assess the prevalence of human immunodeficiency virus (HIV) infection. Following the exclusion of those who were seropositive from the intake sample, a cohort of 138 HIV-negative cocaine abusers was followed for 18 months to assess seroincidence rates and the associated characteristics. HIV status, drug use, sexual risk behaviors, and presence and severity of medical and psychiatric symptoms were collected at intake using a Brazilian version of the Risk Assessment Battery, the Addiction Severity Index (ASI), Symptom Check List-90 (SCL-90), and Beck Depression Inventory (BDI); this was repeated at 6-, 12-, and 18-month intervals.

Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts.

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead.

Proteoglycans on bone marrow endothelial cells bind and present SDF-1 towards hematopoietic progenitor cells.

Recognition events between hematopoietic progenitor cells (HPC) and bone marrow endothelial cells (BMEC) initiate homing of HPC to the bone marrow. The chemokine SDF-1 is present on BMEC and plays a crucial role in bone marrow engraftment. We studied the role of proteoglycans (PGs) on BMEC in binding and presentation of SDF-1.

Mechanism of rat UDP-glucuronosyltransferase 1A6 induction by oltipraz: evidence for a contribution of the Aryl hydrocarbon receptor pathway.

The utility of oltipraz as a cancer chemopreventive agent is thought to depend on the induction of enzymes involved in phase 2 xenobiotic detoxification. Although studies of some enzymes induced by oltipraz implicate a novel transcriptional activating pathway involving Nrf2 and antioxidant-response elements (AREs), the mechanism of phenol UGT induction has remained unclear. Previous work showed that UGT1A6 is transcribed from two promoters, P1 and P2, that are both induced by oltipraz in rat liver.

The targeting of the atToc159 preprotein receptor to the chloroplast outer membrane is mediated by its GTPase domain and is regulated by GTP.

The multimeric translocon at the outer envelope membrane of chloroplasts (Toc) initiates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two Toc GTPases, Toc159 and Toc33/34, mediate preprotein recognition and regulate preprotein translocation. Although these two proteins account for the requirement of GTP hydrolysis for import, the functional significance of GTP binding and hydrolysis by either GTPase has not been defined.

Essential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane.

Two homologous GTP-binding proteins, atToc33 and atToc159, control access of cytosolic precursor proteins to the chloroplast. atToc33 is a constitutive outer chloroplast membrane protein, whereas the precursor receptor atToc159 also exists in a soluble, cytosolic form. This suggests that atToc159 may be able to switch between a soluble and an integral membrane form.

Proteoglycans guide SDF-1-induced migration of hematopoietic progenitor cells.

Stromal cell-derived factor-1 (SDF-1) is a chemoattractant involved in hematopoietic progenitor cell (HPC) trafficking to the bone marrow. We studied the role of bone marrow endothelial proteoglycans (PGs) in SDF-1-mediated migration of HPC using a transwell assay. A subclone of progenitor cell line KG-1 (KG-1v) was used, displaying CXCR4-dependent transmigration.

Homing and clonogenic outgrowth of CD34(+) peripheral blood stem cells: a role for L-selectin?

Objective: After transplantation of hematopoietic stem cells, adhesion molecules play a major role in the multistep process of engraftment in which L-selectin is suggested to be of relevance. A positive correlation previously was found between the number of reinfused L-selectin(+) stem cells and platelet recovery. In the present study, we determined the role of L-selectin in different engraftment steps, i.

Extensive early apoptosis in frozen-thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation.

Stem cell doses necessary for engraftment after myelo-ablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34+ cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed.

Glucuronidation of acetaminophen catalyzed by multiple rat phenol UDP-glucuronosyltransferases.

Gunn rats glucuronidate acetaminophen (APAP) at reduced rates and show increased susceptibility to APAP-induced hepatotoxicity. This defect is presumed to involve UDP-glucuronosyltransferase (UGT) 1A6, which is nonfunctional in Gunn rats, but it is currently unclear whether other 1A family members are also involved. In humans, two 1A isoforms are known to be active (1A6 and 1A9) but 1A6 form has a 25-fold lower apparent K(m) (2 mM).

Protein translocon at the Arabidopsis outer chloroplast membrane.

Chloroplasts are organelles essential for the photoautotrophic growth of plants. Their biogenesis from undifferentiated proplastids is triggered by light and requires the import of hundreds of different precursor proteins from the cytoplasm. Cleavable N-terminal transit sequences target the precursors to the chloroplast where translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes enable their import.

FLU: a negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana.

Tetrapyrroles such as chlorophylls and bacteriochlorophylls play a fundamental role in the energy absorption and transduction activities of photosynthetic organisms. Because of these molecules, however, photosynthetic organisms are also prone to photooxidative damage. They had to evolve highly efficient strategies to control tetrapyrrole biosynthesis and to prevent the accumulation of free intermediates that potentially are extremely destructive when illuminated.

Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane.

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood.

Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells.

Objective: Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC).

An alternative promoter contributes to tissue- and inducer-specific expression of the rat UDP-glucuronosyltransferase 1A6 gene.

UDP-glucuronosyltransferase 1A6 (UGT1A6), a key enzyme catalyzing the glucuronidation of small planar phenols and amines, is expressed in a tissue- and inducer-dependent manner. Expression is high in kidney, gastrointestinal tract, and induced liver, with low expression in spleen, lung, and ovary. Exposure to certain chemicals, such as 3-methylcholanthrene, benzo[a]pyrene, beta-naphthoflavone, and oltipraz elevates UGT1A6 mRNA in liver and to a lesser extent gastrointestinal tract and kidney, but not in other tissues.

Molecular biology of chloroplast biogenesis: gene expression, protein import and intraorganellar sorting.

The chloroplast is the hallmark organelle of plants. It performs photosynthesis and is therefore required for photoautotrophic plant growth. The chloroplast is the most prominent member of a family of related organelles termed plastids which are ubiquitous in plant cells.