1H and 13C solution- and solid-state NMR investigation into wax products from the Fischer-Tropsch process.

(1)H and (13)C solid- and solution-state NMR have been used to characterise waxes produced in the Fischer-Tropsch reaction, using Co-based catalysts either unpromoted or promoted with approximately 1 wt% of either cerium or rhenium. The aim was to measure average structural information at the submolecular level of the hydrocarbon waxes produced, along with identification of the minor products, such as oxygenates and olefins, which are typically observed in these waxes. A parameter of key interest is the average number of carbon atoms within the hydrocarbon chain (N(C)). A wax prepared using an unpromoted Co/Al(2)O(3) catalyst had N(C)∼20, whilst waxes made using rhenium- or cerium-promoted Co/Al(2)O(3) catalysts were found to have N(C)∼21. All three samples contained small amounts of oxygenates and alkenes. The subtle differences found in the waxes, in particular the minor species produced, demonstrate that the different promoters have different effects during the reaction, with the Re-promoted catalyst producing the fewest by-products. It is shown in (13)C solid-state NMR spectra that for that for longer chain (compared to the lengths of chain in previous studies) waxes that the lack of resolution and the complexities added by the differential cross-polarisation (CP) dynamics mean that it is difficult to accurately determine N(C) from this approach. However the N(C) determined by (13)C CP magic angle spinning NMR is broadly consistent with the more accurate solution approaches used and suggest that the wax characteristics do not change in solution. On this basis an alternative approach for determining N(C) is suggested based on (1)H solution state NMR that provides a higher degree of accuracy of the chain length as well as information on the minor constituents.

Oncogenic mutations cause dramatic, qualitative changes in the transcriptional activity of c-Myb.

The v-Myb oncoprotein encoded by Avian Myeloblastosis Virus is highly oncogenic, induces leukemias in chickens and mice and transforms immature hematopoietic cells in vitro. The v-Myb protein is a mutated and truncated version of c-Myb, a DNA-binding transcription factor expressed in many cell types that is essential for normal hematopoiesis. Previous studies suggested that two types of differences, DNA binding domain mutations and the deletion of a C-terminal negative regulatory domain were important for increasing the transforming activity of v-Myb. Here, we combined structure-function studies of the v-Myb and c-Myb proteins with unbiased microarray-based transcription assays to compare the transcriptional specificities of the two proteins. In human cells, the v-Myb and c-Myb proteins displayed strikingly different activities and regulated overlapping, but largely distinct sets of target genes. Each type of mutation that distinguished v-Myb from c-Myb, including the N- and C-terminal deletions, DNA binding domain changes and mutations in the transcriptional activation domain, affected different sets of target genes and contributed to the different activities of c-Myb and v-Myb. The results suggest that v-Myb is not just a de-repressed version of c-Myb. Instead, it is a distinct transcriptional regulator with a unique set of activities.

Analysis of gene transfer efficiency of retrovirus producer cell transplantation for in situ gene transfer to hematopoietic cells.

OBJECTIVE: The aim of this study was to assess the gene transfer efficiency of an in situ administration protocol for hematopoietic stem/progenitor cells in the rhesus macaque (Macaca mulatta) animal model. MATERIALS AND METHODS: Moloney murine leukemia virus amphotropic vector producer cells (1--2 x 10(8) cells/animal) were transplanted into the femoral bone marrow cavities of six macaques. To determine if the levels of gene transfer could be increased, a second injection at the same dose of producer cells was performed into the iliac crest in three of the six macaques. RESULTS: We demonstrated that 0.02-0.1% of peripheral blood mononuclear cells contained the vector transgene for up to 12 months following the initial administration of producer cells. Hematopoietic progenitor cell assays indicated that the neomycin phosphotransferase gene was detected in 10--30% of progenitor cell colonies. A humoral immune response directed toward viral particles was demonstrated in all animals. Additionally, we demonstrated that an increase in the levels of transduced cells, up to 1% of circulating peripheral blood mononuclear cells and granulocytes, contain the transgene following producer cell readministration. CONCLUSIONS: These data demonstrate the successful in situ gene transfer to hematopoietic stem/progenitor cells and circulating peripheral blood mononuclear cells that persists as long as 12 months postinjection, in the absence of any preconditioning.

Civilian spontaneous pneumothorax. Treatment options and long-term results.

The treatment of spontaneous pneumothorax in the civilian population can be influenced by the age of the patient and the presence of associated pulmonary disease. The medical records of 130 patients who presented with 168 occurrences of SP were reviewed during an 11-year period (1973 to 1984). Follow-up was from a minimum of 30 months to 13 years (mean 6.3 years). The therapeutic options included observation alone (40 occurrences), thoracentesis (6 occurrences), chest tube thoracostomy (102 occurrences), and thoracotomy (20 occurrences). Treatment of SP should be prompt with the objective of complete re-expansion of the lung and prevention of recurrent pneumothorax. This should be accomplished by the use of chest tube thoracostomy with early addition of thoracotomy as necessary. Selected use of thoracentesis can be effective. The use of observation alone can be dangerous and is associated with a higher recurrence rate.

Optimization of equine infectious anemia derived vectors for hematopoietic cell lineage gene transfer.

Gene transfer into hematopoietic cells may allow correction of a variety of hematopoietic and metabolic disorders. Optimized HIV-1 based lentiviral vectors have been developed for improved gene transfer and transgene expression into hematopoietic cells. However, the use of HIV-1 based vectors for human gene therapy may be limited due to ethical and biosafety issues. We report that vectors based on the non-primate equine infectious anemia virus (EIAV) transduce a variety of human hematopoietic cell lines and primary blood cells. To investigate optimization of gene expression in hematopoietic cells, we compared a variety of post-transcriptional elements and promoters in the context of EIAV vectors. We observed cell specific increase in the number of transgene expressing cells with the different post-transcriptional elements, whereas the use of elongation factor alpha 1 (EFalpha1) promoter resulted in significant increases in both the number of transgene expressing cells and the level of transgene protein in all cell types tested. We then demonstrate increased transduction of hematopoietic cells using a second-generation EIAV vector containing a self-inactivating EIAV LTR and the EIAV central polypurine tract (cppt). These data suggest that optimized EIAV vectors may be a suitable alternative to HIV-1 vectors for use in hematopoietic gene therapy.

Signal transducer and activator of transcription 3 activates CCAAT enhancer-binding protein delta gene transcription in G0 growth-arrested mouse mammary epithelial cells and in involuting mouse mammary gland.

The CCAAT enhancer-binding protein (C/EBP) family of transcription factors is implicated in the regulation of cell proliferation and differentiation in a variety of tissues. C/EBPdelta is involved in regulating G(0) growth arrest and apoptosis of mouse mammary epithelial cells. This study shows that activation of signal transducer and activator of transcription 3 (Stat3), but not activation of Stat1 or Stat5, occurs concurrently with G(0) growth arrest of HC11 mouse mammary epithelial cells, but not NIH 3T3 fibroblasts. Promoter analysis demonstrates that the C/EBPdelta promoter fragment involved in transcriptional activation during G(0) growth arrest contains a Stat3 binding site and that mutation of this site eliminates the G(0) growth arrest inducibility of the C/EBPdelta promoter. Overexpression of Stat3 increases C/EBPdelta promoter activity during G(0) growth arrest of HC11 cells, whereas dominant negative Stat3 decreases C/EBPdelta promoter activity under the same conditions. Neither Stat3 overexpression nor dominant negative Stat3 expression influences C/EBPdelta promoter activity in growing HC11 cells or G(0) growth-arrested NIH3T3 cells, demonstrating that the effect is specific to G(0) growth arrest of mammary epithelial cells. Band shift assays and antibody interference assays demonstrate specific binding of Stat3 to the acute phase response element in the C/EBPdelta promoter in G(0) growth-arrested HC11 cell extracts and 24 h involuting mouse mammary gland extracts. These data indicate that Stat3 activates C/EBPdelta transcription in G(0) growth-arrested mouse mammary epithelial cells and binds to the C/EBPdelta promoter during involution. An autocrine mechanism of Stat3 activation is proposed.

Transcriptional regulation of C/EBPdelta in G(0) growth-arrested mouse mammary epithelial cells.

Little is known about the control of cell cycle exit/G(0) entry, or the regulation of genes that are expressed during G(0). In this report we used primer extension analysis to demonstrate the high level of C/EBPdelta mRNA expression in G(0) growth-arrested HC11 mouse mammary epithelial cells and to identify the C/EBPdelta transcription start site. The C/EBPdelta gene transcription rate and promoter activity are both highly induced in G(0) growth-arrested HC11 cells. The C/EBPdelta gene promoter also exhibits G(0)-specific autoregulation. In contrast, the C/EBPdelta promoter activity decreases in G(0) growth-arrested NIH 3T3 cells. These data indicate that C/EBPdelta is among a relatively small number of genes actively transcribed during G(0) growth arrest. C/EBPdelta may regulate the expression of genes implicated in the initiation or maintenance of mammary epithelial cell G(0) growth arrest.

CCAAT/enhancer-binding protein delta regulates mammary epithelial cell G0 growth arrest and apoptosis.

CCAAT/enhancer-binding proteins (C/EBPs) are a highly conserved family of DNA-binding proteins that regulate cell-specific growth, differentiation, and apoptosis. Here, we show that induction of C/EBPdelta gene expression during G0 growth arrest is a general property of mammary-derived cell lines. C/EBPdelta is not induced during G0 growth arrest in 3T3 or IEC18 cells. C/EBPdelta induction is G0-specific in mouse mammary epithelial cells; C/EBPdelta gene expression is not induced by growth arrest in the G1, S, or G2 phase of the cell cycle. C/EBPdelta antisense-expressing cells (AS1 cells) maintain elevated cyclin D1 and phosphorylated retinoblastoma protein levels and exhibit delayed G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. Conversely, C/EBPdelta-overexpressing cells exhibited a rapid decline in cyclin D1 and phosphorylated retinoblastoma protein levels, a rapid increase in the cyclin-dependent kinase inhibitor p27, and accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. When C/EBPdelta levels were rescued in AS1 cells by transfection with a C/EBPdelta "sense" construct, normal G0 growth arrest and apoptosis were restored. These results demonstrate that C/EBPdelta plays a key role in the regulation of G0 growth arrest and apoptosis in mammary epithelial cells.

Repression of tax-mediated human t-lymphotropic virus type 1 transcription by inducible cAMP early repressor (ICER) protein in peripheral blood mononuclear cells.

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia and is characterized by long periods of clinical latency with low levels of viral production. Transcription of HTLV-1 is controlled through sequences in the promoter and enhancer regions of the long terminal repeat of the integrated provirus. Important among these sequences are three 21 bp imperfect repeats responsive to the viral oncogenic protein Tax (TRE). Members of the CREB/ATF-1/CREM family of transcription factors bind to TRE-1 and are critical for HTLV-1 transcription. Other less studied family members include the inducible cAMP early repressor (ICER) proteins. ICER proteins lack phosphorylation and activation domains and are potent inhibitors of transcription. The ability of ICER to bind TRE-1 and its effects on HTLV-1 Tax mediated transcription have not been studied in the natural cell targets of the virus, peripheral blood mononuclear cells (PBMC). We show that ICER mRNA levels are low in quiescent PBMC, but rise and remain elevated for up to 18 hr after mitogenic stimulation of these cells. Electrophoretic mobility shift assays using recombinant Tax and ICER demonstrate that ICER binds TRE-1 and that binding is increased in the presence of Tax. Furthermore, over expression of ICER IIgamma suppressed Tax-mediated transcription whereas an anti-sense ICER II plasmid designed to block endogenous ICER enhanced Tax-mediated transcription in activated PBMC. Together our data indicate that ICER inhibits Tax-mediated transcription in activated PBMC and suggest a role for ICER in maintenance of HTLV-1 persistence.

Comparison of gene transfer efficiencies and gene expression levels achieved with equine infectious anemia virus- and human immunodeficiency virus type 1-derived lentivirus vectors.

This report compares gene transfer efficiencies as well as durations and levels of gene expression for human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) lentiviral vectors in a variety of human cell types in vitro. EIAV and HIV vectors transduced equivalent numbers of proliferating and G1/S- and G2/M-arrested cells, and both had very low efficiencies of transduction into G0-arrested cells. Analysis of the levels of both the enhanced green fluorescent protein (EGFP) and mRNA demonstrated that the HIV-transduced cells expressed greater levels of EGFP protein and RNA than the EIAV-transduced cells. Measurements of vector-derived EGFP RNA half-lives were fourfold higher with the HIV vector than with the EIAV vector. Long-term culture of EIAV-transduced human cells showed a significant decrease in the number of cells expressing the transgene; however, no corresponding loss was found in EIAV-transduced equine cells. In contrast, only a moderate decrease in the number of transgene-expressing cells was seen with the HIV vectors. Taken together, these results demonstrate that the EIAV vectors transduced human cells with efficiencies similar to those of the HIV vectors. However, our data indicate that transgene expression from EIAV vectors is limited by the instability of vector-derived RNA transcripts and silencing of the EIAV vectors over time.

Human T-cell lymphotropic virus type 1 Tax mediates enhanced transcription in CD4+ T lymphocytes.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma and is associated with a variety of immunoregulatory disorders. HTLV-1 has been shown to bind to and infect a variety of hematopoietic and nonhematopoietic cells. However, both in vivo and in vitro, the provirus is mostly detected in and preferentially transforms CD4+ T cells. The molecular mechanism that determines the CD4+ T-cell tropism of HTLV-1 has not been determined. Using cocultures of purified CD4+ and CD8+ T cells with an HTLV-1 producing cell line, we measured viral transcription by using Northern (RNA) blot analysis, protein production by using a p24 antigen capture assay and flow cytometric analysis for viral envelope, and proviral integration by using DNA slot blot analysis. We further measured HTLV-1 long terminal repeat-directed transcription in purified CD4+ and CD8+ T cells by using transient transfection assays and in vitro transcription. We demonstrate a higher rate of viral transcription in primary CD4+ T cells than in CD8+ T cells. HTLV-1 protein production was 5- to 25-fold greater in CD4+ cocultures and mRNA levels were 5-fold greater in these cultures than in the CD8+ cocultures. Transient transfection and in vitro transcription indicated a modest increase in basal transcription in CD4+ T cells, whereas there was a 20-fold increase in reporter gene activity in CD4+ T cells cotransfected with tax. These data suggest that unique or activated transcription factors, particularly Tax-responsive factors in CD4+ T cells, recognize regulatory sequences within the HTLV-1 long terminal repeat, and this mediates the observed enhanced viral transcription and ultimately the cell tropism and leukemogenic potential of the virus.

Analysis of gene transfer and expression in skeletal muscle using enhanced EIAV lentivirus vectors.

Skeletal muscle is an attractive target tissue for gene therapy involving both muscle and nonmuscle disorders. HIV-1-based vectors transduce mature skeletal muscle; however, the use of these vectors for human gene therapy may be limited by biosafety concerns. In this study, we investigated gene transfer using lentivirus vectors based on the equine infectious anemia virus (EIAV) in skeletal muscle in vitro and in vivo. EIAV vectors transduce proliferating and differentiating C2C12 mouse muscle cells; furthermore, the addition of the woodchuck hepatitis posttranscriptional element to EIAV vectors markedly increases gene expression in these cells. A single injection of EIAV vectors into skeletal muscle of adult mice led to detectable gene marking and gene expression for the duration of the 3-month study. Use of a second-generation EIAV self-inactivating vector (E-SIN) increased transduction in muscle cells in vitro, and injection of E-SIN vectors into skeletal muscle resulted in increased gene marking and gene expression compared to first-generation EIAV vectors.

Comparison of HTLV-I basal transcription and expression of CREB/ATF-1/CREM family members in peripheral blood mononuclear cells and Jurkat T cells.

HTLV-I is the etiologic agent of adult T-cell leukemia/lymphoma and is associated with tropical spastic paraparesis/HTLV-I-associated myelopathy. Following integration into the host cell genome, HTLV-I replication is regulated by both host and viral mechanisms that control transcription. Low levels of viral transcription (basal transcription) occur before expression of the virally encoded Tax protein (Tax-mediated transcription). Members of the cyclic adenosine monophosphate (cAMP) response element binding (CREB)/activating transcription factor 1 (ATF-1) family of transcription factors bind three 21-bp repeats (Tax-responsive element-1, or TRE-1) within the viral promoter and are important for basal and Tax-mediated transcription. Using mitogen stimulated and quiescent peripheral blood mononuclear cells (PBMC) and Jurkat cells, we compared differences in basal transcription and amounts and binding of transcription factors with TRE-1. We demonstrate that amounts of transcriptionally active phosphorylated CREB protein (P-CREB) differ between activated PBMC and Jurkat cells. Following stimulation, P-CREB levels remain elevated in PBMC for up to 24 hours whereas CREB is dephosphorylated in Jurkat cells within 4 hours following stimulation. The differences in P-CREB levels between PBMC and Jurkat cells were directly correlated with basal transcription of HTLV-I in the two cell types. Using electrophoretic mobility shift assays, we determined that the pattern of band migration differed between the two cell types. These data demonstrate that PBMC differentially regulate basal HTLV-I transcription compared with Jurkat T cells, and this differential regulation is due, in part to differential phosphorylation and binding of CREB/ATF-1 to TRE-1 in the HTLV-I promoter. We demonstrate the utility of using primary lymphocyte models to study HTLV-I transcription in the context of cell signaling and suggest that activated PBMC maintain elevated levels of P-CREB, which promote basal HTLV-I transcription and enhance viral persistence in vivo.

Rhesus monkey model for fetal gene transfer: studies with retroviral- based vector systems.

Many life-threatening conditions that can be diagnosed early in gestation may be treatable in utero using gene therapy. In order to determine in utero gene transfer efficiency and safety, studies were conducted with fetal rhesus monkeys as a model for the human. Included in these studies were Moloney murine leukemia virus (MLV)-based amphotropic retrovirus, vesicular stomatitis virus-G (VSV-G) pseudotyped MLV, and a VSV-G pseudotyped HIV-1-based vector, all expressing the enhanced green fluorescent protein (EGFP) as a reporter gene and driven by a cytomegalovirus-immediate early promoter (N = 16). Rhesus monkey fetuses were administered viral vector supernatant preparations by the intraperitoneal (ip) (N = 14) or intrahepatic (ih) (N = 2) routes via ultrasound guidance at 55 +/- 5 days gestation (late first trimester; term 165 +/- 10 days). Fetuses were monitored sonographically, specimens were collected prenatally and postnatally, and tissue harvests were performed at birth or 3 or 6 months postnatal age (3-10 months post-gene transfer). PCR analyses demonstrated that transduced cells were present at approximately 1.2% in peripheral blood mononuclear cells from fetuses administered amphotropic MLV, <0.5% in fetuses receiving MLV/VSV-G, and approximately 4.2% for the lentiviral vector, which decreased to 2% at birth. Hematopoietic progenitors showed that overall (mean of all time points assessed), approximately 25% of the collected colonies were positive for the EGFP transgene with the lentiviral vector, which was significantly greater than results achieved with the MLV-based vector systems (4-9%; P < or = 0.001-0.016). At necropsy, 0.001-10% of the total genomic DNA was positive for EGFP in most tissues for all groups. EGFP-positive fluorescent cells were found in cell suspensions of thymus, liver, spleen, lymph nodes, cerebral cortex, and bone marrow (0.5-6%). Overall, the results of these studies have shown: (1) healthy infants expressing vector sequences up to 10 months post-gene transfer, (2) fetal primate administration of retroviral vectors results in gene transfer to multiple organ systems, (3) the highest level of gene transfer to hematopoietic progenitors was observed with the lentiviral vector system, and (4) there was no evidence of transplacental transfer of vector sequences into the dams. The rhesus monkey is an important preclinical primate model system for exploring gene transfer approaches for future applications in humans.