BACKGROUND: We have developed a novel system for expansion of gene-modified hematopoietic stem/progenitor cells to overcome the low efficiency of current gene transfer methodology. This system involves 'selective amplifier genes', that encode fusion proteins between the granulocyte colony-stimulating factor receptor (GCR) and the hormone-binding domain of estrogen receptor (ER). Hematopoietic progenitors expressing the chimeras showed estrogen-responsive growth in a controllable manner. However, endogenous estrogen may activate the fusion proteins in vivo, depending on the hormonal status of the subjects. METHODS: We replaced ER with a mutant receptor (TmR) which specifically binds to 4-hydroxytamoxifen (Tm), to overcome limitations with wild-type ER. Interleukin-3 (IL-3)-dependent Ba/F3 cells and hematopoietic progenitor cells transduced with the resultant fusion proteins (GCRTmR and delta GCRTmR) were examined for ligand-inducible growth. RESULTS: GCRTmR- and delta GCRTmR-expressing Ba/F3 showed IL-3-independent growth in response to Tm, while the cells were unresponsive to estrogen at concentrations up to 10(-7)-10(-6) M. Furthermore, murine bone marrow cells transduced with GCRTmR and delta GCRTmR formed colonies in methyl-cellulose medium in response to Tm, while virtually no colonies appeared with 10(-7) M estrogen or without cytokines. CONCLUSIONS: These results suggest that influences of the endogenous estrogen can be almost eliminated by using the GCRTmR/Tm or delta GCRTmR/Tm system to expand gene-modified hematopoietic stem/progenitor cells.

Animals↗

PubMedDate not supplied

Realities of high-throughput liquid chromatography/mass spectrometry purification of large combinatorial libraries: a report on overall sample throughput using parallel purification.

We report on the development of a validated, streamlined high-throughput process for the purification of parallel-synthesis-derived combinatorial libraries. The steps involved in this library purification process include dissolution of dry films of crude synthetic material, dual-column LC/MS purification, dual-column postpurification analysis, quantitation, reformatting, and submission of pure compounds for registration. Although the purification and postpurification analysis times decreased essentially linearly as a function of the number of HPLC columns employed, it was not possible to decrease the total purification process time linearly as a function of the number of columns employed in the system. This was due primarily to the fact that numerous steps in the total purification process are independent of sample analysis and purification (e.g., evaporation, reconstitution, and reformatting, etc.). Additionally, experiments were also performed to assess whether separate gradient pumps were necessary for each channel of this two-channel LC/MS or if acceptable results could be reliably obtained by splitting the flow from one set of gradient pumps between two HPLC columns. On the basis of the parallel, two-column LC/MS system employed in this work, throughput estimates were extrapolated to more massively parallel systems (e.g., four-channel LC/MS).

Amino Acids↗

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Unwinding and hydrodynamic flow linear dichroism characteristics of supercoiled DNA covalently modified with two isomeric methylchrysene diol epoxides of different biological activities.

Adducts derived from the covalent binding of two positional monomethyl-substituted isomers of a bay region chrysene diol epoxide to supercoiled pIBI30 DNA (2926 base pairs/genome) were prepared, and their characteristics were investigated by a combination of gel electrophoresis and flow linear dichroism techniques. The 5- and 6-methyl derivatives of trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene [(+)-5- and (+)-6-MeCDE, respectively], both with 1R,2S,3S,4R stereochemistry, are characterized by significant differences in their biological activities [Melikian et al. (1988) Cancer Res. 48, 1781-1787]. When covalently bound to plasmid DNA, these two molecules give rise to striking differences in the gel electrophoretic and flow hydrodynamic characteristics of the modified supercoiled DNA. The hydrodynamic flow linear dichroism of linearized DNA molecules (obtained by EcoRI enzyme digestion of covalently closed supercoiled pIBI30 DNA), modified covalently with the highly tumorigenic and mutagenic (+)-5-MeCDE derivative, indicates that flexible joints, bends, or kinks are formed at the site of binding of (+)-5-MeCDE. Slab gel data, as well as ethidium bromide-titration tube agarose gel electrophoresis data, indicate that the formation of (+)-5-MeCDE-DNA lesions causes the removal of superhelical turns with an unwinding angle of 13 +/- 3 degrees per covalently bound polycyclic aromatic residue. In contrast, the biological inactive (+)-6-MeCDE does not significantly alter the characteristics of supercoiled DNA, the unwinding angle is only 2.7 +/- 1 degrees, and the changes in persistence lengths detected by the flow linear dichroism technique are significantly smaller than in the case of (+)-5-MeCDE-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗

PubMedDate not supplied

Base sequence-dependent bends in site-specific benzo[a]pyrene diol epoxide-modified oligonucleotide duplexes.

The site specifically modified oligonucleotides 5'-d(TCCTCCTG1G2CCTCTC) (I) and 5'-d(CTATG1G2G3TATC) (II) were synthesized with single modified guanine residues at positions G1, G2, or G3, derived from the covalent binding reaction of 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE) with the exocyclic amino groups of the guanine residues. In denaturing 20% polyacrylamide gels, the electrophoretic mobilities of the (+)-anti-BPDE-modified oligonucleotides I and II are slower than the mobilities of the respective unmodified oligonucleotides and independent of the positions of the BPDE-modified guanines. However, in the double-stranded forms in native 8% polyacrylamide gels, the electrophoretic mobilities of the duplexes with lesions at G2 or G3 are remarkably slower (reductions in mobilities up to approximately 40%) than to duplexes with lesions at G1 and are attributed to physical bends or flexible hinge joints at the sites of the BPDE lesions. These sequence-dependent mobility effects occur whenever the BPDE-modified guanine residues with (+)-trans-stereochemistry are flanked by unmodified G's on the 5'-side. These retarded electrophoretic mobilities are attributed to bending induced by steric hindrance effects involving the bulky 5'-flanking guanines and the pyrenyl residues that are known to point into the 5'-direction relative to the modified G [Cosman, M., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918]. These anomalous electrophoretic mobility effects are not observed in the case of (-)-anti-BPDE-modified sequences I with trans-(-)-anti-BPDE-N2-dG adduct stereochemistry.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMedDate not supplied

Growth factor practice patterns among pediatric oncologists: results of a 1998 Pediatric Oncology Group Survey. Economic Evaluation Working Group the Pediatric Oncology Group.

The American Society of Clinical Oncology (ASCO) guidelines on growth factor (GF) use recommend applying adult-derived guidelines in pediatric oncology. An ASCO survey of adult oncology GF use determined the preference for first degree prophylaxis (use of GF when febrile neutropenia [FN] is expected to be high in untreated patients), second-degree prophylaxis (administration of GF after a documented episode of FN on a previous cycle of chemotherapy), and intervention in the treatment of FN. Similar preferences have not been evaluated in pediatrics. The purpose of this study was to (1) characterize GF use in pediatric oncology; (2) correlate use patterns with demographic factors; and (3) compare the Pediatric Oncology Group (POG) and ASCO surveys. The ASCO survey was revised for use within pediatric oncology and was mailed to the physician membership of POG; 341 were returned (86% completion rate). Comparisons were made with the ASCO survey. Most (76%) physicians said GF use was determined by protocol requirements and most (70%) patients were entered on POG protocols. GF use as first-degree prophylaxis was selected 40% of the time, which was significantly greater than in adults; this was most influenced by anticipated duration of neutropenia (> or =7 days). The severity of the initial clinical course (e.g., neutropenia, infection) influenced use in second-degree prophylaxis; dose reduction alone was never selected. For FN, GF use was 45%, with lower preferences in uncomplicated FN (16%-38%) compared with complicated FN (66%). POG respondents endorse greater use of GF for first-and second-degree prophylaxis but less use in uncomplicated FN than do ASCO respondents. These patterns may reflect different strategies, including the role of chemotherapy, value of dose intensity, and perceived toxicity of regimens. Given these differences, adult-based guidelines may not be appropriate for pediatrics.

Age Factors↗

PubMedDate not supplied

Application of a slow-release fertilizer for oil bioremediation in beach sediment.

A 105-d field experiment was conducted to determine the potential of the slow-release fertilizer, Osmocote (Scotts, Marysville, OH), to stimulate the indigenous microbial biodegradation of petroleum hydrocarbons in an oil-spiked beach sediment on an intertidal foreshore in Singapore. Triplicate microcosms containing 80 kg of weathered sediment, spiked with 5% (w/w) Arabian light crude oil and 1.2% (w/w) Osmocote pellets, were established, together with control microcosms minus Osmocote. Relative to the control, the presence of the Osmocote sustained a significantly higher level of nutrients (NH(4)(+)-N, NO(3)(-)-N, and PO(4)(3-)-P) in the sediment pore water over the duration of the experiment. The metabolic activity of the indigenous microbial biomass, as measured using an intracellular dehydrogenase enzyme assay, was also significantly enhanced over the duration of the experiment in amended sediments. The loss of total recoverable petroleum hydrocarbons (TRPH) and biodegradation of total n-alkanes (C(10)-C(33)), branched alkanes (pristane and phytane), as well as total target polycyclic aromatic hydrocarbons (PAHs) (two- to six-ring), in both the control and Osmocote-amended sediments, followed a first-order biodegradation model. The first-order loss rate of total recoverable petroleum hydrocarbons was 2.57 times greater than that of the control. The hopane-normalized rate constants for total n-alkane, branched alkane, and total target PAH biodegradation in the Osmocote-treated sediments were 3.95-, 5.50-, and 2.45-fold higher than the control, respectively. Overall, the presence of Osmocote was able to significantly enhance and accelerate the biodegradation of aliphatics and PAHs in oil-contaminated sediments under natural field conditions in an intertidal foreshore environment.

Biodegradation, Environmental↗

PubMedDate not supplied

Bone-graft harvesting from iliac and fibular donor sites: techniques and complications.

The ilium and the fibula are the most common sites for bone-graft harvesting. The different methods for harvesting iliac bone graft include curettage, trapdoor or splitting techniques for cancellous bone, and the subcrestal-window technique for bicortical graft. A tricortical graft from the anterior ilium should be taken at least 3 cm posterior to the anterior superior iliac spine (ASIS). Iliac donor-site complications include pain, neurovascular injury, avulsion fractures of the ASIS, hematoma, infection, herniation of abdominal contents, gait disturbance, cosmetic deformity, violation of the sacroiliac joint, and ureteral injury. The neurovascular structures at risk for injury during iliac bone-graft harvesting include the lateral femoral cutaneous, iliohypogastric, and ilioinguinal nerves anteriorly and the superior cluneal nerves and superior gluteal neurovascular bundle posteriorly. Violation of the sacroiliac joint can be avoided by limiting the harvested area to 4 cm from the posterior superior iliac spine (PSIS) and by not penetrating the inner cortex. The caudal limit for bone harvesting should be the inferior margin of the roughened area anterior to the PSIS on the outer table to keep from injuring the superior gluteal artery. Potential complications of fibular graft harvesting include neurovascular injury, compartment syndrome, extensor hallucis longus weakness, and ankle instability. The neurovascular structures at risk for injury during fibular bone-graft harvesting include the peroneal nerves and their muscular branches in the proximal third of the fibular shaft and the peroneal vessels in the middle third.

Bone Transplantation↗