IntroductionAdhesive interactions between hematopoietic progenitor and stem cells and the hematopoietic microenvironment play a critical role in maintaining hematopoiesis. [1][2][3][4][5] Hematopoietic growth factors are potent regulators of hematopoiesis. In addition, these proteins have been implicated in modulating adhesion between hematopoietic progenitor cells and extracellular matrix proteins via changes in integrin receptor activation. [6][7][8][9] The role of adhesion molecules alone or with growth factors in maintaining proliferation, differentiation, and survival of hematopoietic cells is less understood, 10,11 but collaboration between growth factor receptors and integrins has been hypothesized to be necessary for normal hematopoietic development. 12 Specifically, integrin receptors such as alpha 4 beta 1 (␣ 4 ␤ 1 ) and/or alpha 5 beta 1 (␣ 5 ␤ 1 ), may collaborate in unique ways with receptor tyrosine kinases, to influence cellular events. In this regard, mice deficient in the receptor tyrosine kinase, c-Kit, its ligand stem cell factor (SCF), or ␤ 1 integrins demonstrate hematopoietic defects of varying severity, suggesting critical roles for these proteins in normal blood development. 1,13,14 Our laboratory and other investigators have shown that receptors of the extracellular matrix protein, fibronectin (FN), are involved in the adhesion of hematopoietic cells, including stem and progenitor cells in the hematopoietic microenvironment. [15][16][17][18][19][20] FN is expressed at high levels throughout the hematopoietic microenvironment. 21,22 The FN molecule contains binding sites for heparin, collagen, fibrin, and gelatin, suggesting that it plays an important role in regulating the architecture of the hematopoietic microenvironment. The binding of hematopoietic cells to FN is mediated by at least 2 integrin receptors. The ␣ 5 ␤ 1 receptor recognizes the minimal binding sequence Arg-Gly-Asp (single-letter amino acid code: RGD), as well as 2 other synergistic binding sites, all of which are located within the cell-binding domain of the FN molecule, 23,24 and ␣ 4 ␤ 1 binds sequences within the alternatively spliced IIICS region of FN defined by the synthetic peptides CS-1 and CS-5. 25,26 These receptors play a critical role in normal hematopoietic development. 1,27,28 Mutant mice homozygous for null mutations of c-Kit, or its ligand SCF, die in embryonic development or shortly after birth due to severe anemia. 14,[29][30][31] Viable homozygous mutants of c-Kit also demonstrate severe anemia and a marked reduction in both immature and mature erythroid progenitors. Data from these mutant mice show a critical role for c-Kit-mediated signaling in normal erythroid development. 29,30 Interactions of erythroid cells with FN are also believed to be essential for erythropoiesis, particularly for terminal stages of erythroid differentiation. [32][33][34][35][36] Erythroid progenitors express both ␣ 4 ␤ 1 and ␣ 5 ␤ 1 . 37,38 Efficient production of mature cells in vitro requires adhesion to FN in some systems....

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To improve the purity of lentiviral vector supernatants for clinical studies we have evaluated plasmid DNA removal from lentiviral vectors and also the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol. Optimal conditions of plasmid DNA removal by benzonase treatment were established by varying the temperature, time, and benzonase concentrations in the reaction mix and were determined to be 50 units of benzonase per milliliter of vector supernatant at 37 degrees C, for 15 min. No plasmid DNA was detected, suggesting efficient plasmid degradation was achieved under these experimental conditions. The infectious titer of benzonase-treated lentiviral vector (RRL-CMV-GFP) was nearly identical to the titer of untreated vector (2.3 +/- 0.3 x 10(6) transduction units per milliliter (TU/ml) and 2.7 +/- 0.3 x 10(6) TU/ml, respectively). Analysis of plasmid DNA in concentrated lentiviral vectors shows that concentration substantially decreases the amount of DNA per TU. Analysis of the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol suggests that a minimal amount of plasmid is transferred to transduced cells if the vector supernatant was not previously treated with benzonase. In conclusion, benzonase treatment is effective in eliminating plasmid DNA from vector supernatants and treatment does not affect infectious titers. However, because there is minimal transfer of plasmid DNA to transduced cells under ex vivo transduction conditions, DNA removal from lentiviral vectors may not be essential for all ex vivo clinical applications.

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.

Endorheic lakes of the northern Great Plains encompass a wide range of environmental parameters (e.g., salinity, pH, DOC, Ca, nutrients, depth) that vary 1000-fold among sites and through the past 2000 years due to variation in basin hydrology and evaporative forcing. However, while many environmental parameters are known to individually influence zooplankton diversity and taxonomic composition, relatively little is known of the hierarchical relationships among potential controls or of how regulatory mechanisms may change in response to climate variation on diverse scales. To address these issues, we surveyed 70 lakes within a 100 000 km 2 prairie region to simulate the magnitude of environmental change expected to occur over 100-1000 years and to quantify the unique and interactive effects of diverse environmental parameters in regulating pelagic invertebrate community structure at that scale. Multivariate analyses showed that salinity was the principal correlate of changes in invertebrate composition among lakes, with a sequential loss of taxa between salinities of 4 and 50 g total dissolved solids L À1 until one to two species predominated in highly saline systems. In contrast, changes in the concentrations of Ca 2 1 and other mineral nutrients exerted secondary controls of invertebrate assemblages independent of salinity, whereas lake depth provided a tertiary regulatory mechanism structuring species composition. In contrast to these large-scale hierarchical patterns, seasonal surveys (May, July, September) of a subset of 21 lakes in each of 2003-2005 revealed that annual meteorological variation had no measurable effect on pelagic invertebrates, despite large differences in temperature, precipitation, and evaporation arising from regional droughts. Together these findings show that pelagic invertebrate communities in saline lakes are resilient to interannual variability in climate, but suggest that lakes of the northern Great Plains may provide a sensitive model to forecast centennial effects of future climate change.

Parvovirus B19 infection leads to transient aplastic crises in individuals with chronic hemolytic anemias or immunodeficiency states. An additional unexplained sequela of B19 infection is thrombocytopenia. Because B19 is known to have a remarkable tropism for human erythropoietic elements, and is not known to replicate in nonerythroid cells, the etiology of this thrombocytopenia is uncertain. We sought to define the pathobiology of B19-associated thrombocytopenia by examining the role of B19 on in vitro megakaryocytopoiesis. B19 infection of normal human bone marrow cells significantly suppressed megakaryocyte (MK) colony formation compared with mock-infected cells. No such inhibition was observed with a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The B19-MK cell interaction was also studied at the molecular level. Whereas low-density bone marrow cells containing erythroid precursor cells supported B19 DNA replication, no viral DNA replication was observed in B19-infected MK-enriched fractions as determined by the presence of viral DNA replicative intermediates on Southern blots. However, analysis of total cytoplasmic RNA isolated from B19-infected MK fractions showed a low-level expression of the B19 genome as detected by quantitative RNA dot blots as well as by Northern analysis. Furthermore, a frame-shift mutation in a recombinant AAV-B19 hybrid genome segment that encodes the viral nonstructural (NS1) protein significantly reduced the observed inhibition of MK colony formation. These studies indicate tissue- tropism of B19 beyond the erythroid progenitor cell, and lend support to the hypothesis that B19 genome expression may be toxic to cell populations that are nonpermissive for viral DNA replication.

Exposure to inorganic arsenic in utero in C3H mice produces hepatocellular carcinoma in male offspring when they reach adulthood. To help define the molecular events associated with the fetal onset of arsenic hepatocarcinogenesis, pregnant C3H mice were given drinking water containing 0 (control) or 85 ppm arsenic from day 8 to 18 of gestation. At the end of the arsenic exposure period, male fetal livers were removed and RNA isolated for microarray analysis using 22K oligo chips. Arsenic exposure in utero produced significant (p<0.001) alterations in expression of 187 genes, with approximately 25% of aberrantly expressed genes related to either estrogen signaling or steroid metabolism. Real-time RT-PCR on selected genes confirmed these changes. Various genes controlled by estrogen, including X-inactive-specific transcript, anterior gradient-2, trefoil factor-1, CRP-ductin, ghrelin, and small proline-rich protein-2A, were dramatically over-expressed. Estrogen-regulated genes including cytokeratin 1-19 and Cyp2a4 were over-expressed, although Cyp3a25 was suppressed. Several genes involved with steroid metabolism also showed remarkable expression changes, including increased expression of 17beta-hydroxysteroid dehydrogenase-7 (HSD17beta7; involved in estradiol production) and decreased expression of HSD17beta5 (involved in testosterone production). The expression of key genes important in methionine metabolism, such as methionine adenosyltransferase-1a, betaine-homocysteine methyltransferase and thioether S-methyltransferase, were suppressed. Thus, exposure of mouse fetus to inorganic arsenic during a critical period in development significantly alters the expression of various genes encoding estrogen signaling and steroid or methionine metabolism. These alterations could disrupt genetic programming at the very early life stage, which could impact tumor formation much later in adulthood.