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Q Liu

Publications and source records attributed to Q Liu.

At least 19 recordsLinked to original sources
PubMed1997

Mutation of gene-proximal regulatory elements disrupts human epsilon-, gamma-, and beta-globin expression in yeast artificial chromosome transgenic mice.

Previous studies have defined transcriptional control elements, in addition to the promoters, that both lie near individual human beta-globin locus genes and have been implicated in their differential stage-specific regulation during development (i.e., are believed to directly participate in hemoglobin switching). We have reinvestigated the activities during erythropoiesis that might be conferred by two of the more intensively analyzed of these elements, the epsilon-globin gene 5' silencer and the beta-globin gene 3' enhancer, by deleting them from a yeast artificial chromosome that spans the human beta-globin locus, and then analyzing transgenic mice for expression of all of the human genes. These studies show that sequences within the epsilon-globin "silencer" are not only required for silencing but are also required for activation of epsilon-globin transcription; furthermore, deletion of the silencer simultaneously reduced gamma-globin transcription during the yolk sac stage of erythroid development. Analysis of the adult beta-globin gene 3' enhancer deletion showed that its deletion affects only that gene.

Animals↗
PubMed1997

Molecular cloning and chromosomal localization in human and mouse of the SH2-containing inositol phosphatase, INPP5D (SHIP). Amgen EST Program.

The action of lipid phosphatases on inositol phosphates is thought to be one method that the cell uses to attenuate growth factor- or cytokine-induced mobilization of calcium. We have cloned a cDNA from a mouse spleen library that is virtually identical to the recently described inositol tetraphosphate and phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase, SHIP. Chromosomal localization studies in human and mouse by FISH mapping and interspecies backcrossing in mice have demonstrated that human SHIP is localized to 2q36-q37.1 and that mouse SHIP is localized to 1C5.

Amino Acid Sequence↗
PubMed1997

mu-Opioid receptor-stimulated guanosine-5'-O-(gamma-thio)-triphosphate binding in rat thalamus and cultured cell lines: signal transduction mechanisms underlying agonist efficacy.

G protein activation by different mu-selective opioid agonists was examined in rat thalamus, SK-N-SH cells, and mu-opioid receptor-transfected mMOR-CHO cells using agonist-stimulated guanosine-5'-O-(gamma-thio)-triphosphate ([35S]GTP gamma S) binding to membranes in the presence of excess GDP. [D-Ala2, N-MePhe4, Gly5-ol]Enkephalin (DAMGO) was the most efficacious agonist in rat thalamus and SK-N-SH cells, followed by (in rank order) fentanyl = morphine > > buprenorphine. In mMOR-CHO cells expressing a high density of mu receptors, no differences were observed among DAMGO, morphine or fentanyl, but these agonists were more efficacious than buprenorphine, which was more efficacious than levallorphan. In all three systems, efficacy differences were magnified by increasing GDP concentrations, indicating that the activity state of G proteins can affect agonist efficacy. Scatchard analysis of net agon stimulated [35S]GTP gamma S binding revealed two major components responsible for agonist efficacy differences. First, differences in the KD values of agonist-stimulated [35S]GTP gamma S binding between high efficacy agonists (DAMGO, fentanyl, and morphine) and classic partial agonists (buprenorphine and levallorphan) were observed in all three systems. Second, differences in the Bmax value of agonist-stimulated [35S]GTP gamma S binding were observed between DAMGO and morphine or fentanyl in rat thalamus and SK-N-SH cells and between the high efficacy agonists and buprenorphine or levallorphan in all three systems. These results suggest that mu-opioid agonist efficacy is determined by the magnitude of the receptor-mediated affinity shift in the binding of GTP (or[35S]GTP gamma S) versus GDP to the G protein and by the number of G proteins activated per occupied receptor.

Animals↗
PubMed1997

Regulated processing of hepatitis C virus core protein is linked to subcellular localization.

Posttranslational processing and subcellular localization of the HCV core protein are critical steps involved in the assembly of hepatitis C virus (HCV). In this study, both of these events were investigated by in vitro translation and transient COS-1 cell transfection of core protein expression constructs. Mutations at amino acid residues 173 to 174 and 191 to 192 disrupted processing events at the two putative cleavage sites in the C-terminal hydrophobic region of the core protein, indicating that these residues are implicated in the pathway of core protein maturation. As a result, two forms of core protein, C173 and C191, were detected by immunoblotting. Indirect immunofluorescence experiments showed that core proteins C173 and C191, when produced from HCV full-length protein or various polyprotein precursors, displayed a cytoplasmic localization. The C173 species, however, was translocated to the nucleus when expressed in the absence of C191. These findings indicate that preferential cleavage may occur during core protein maturation and that the association of the C191 with the C173 species may contribute to the distinct subcellular distribution of core protein. This may provide a possible mechanism for the control of the diverse biological functions of core protein during HCV replication and assembly.

Amino Acid Sequence↗
PubMed1997

Regulation of myeloid growth and differentiation by the insulin-like growth factor I receptor.

Flow cytometry was used to examine the expression of type I insulin-like growth factor receptors (IGF-IR) on three types of human hematopoietic cells that represent different stages of myeloid lineage development. Both HL-60 (promyeloid) and U-937 (monocytic) cells express abundant IGF-IR protein (> 79% cells positive for the IGF-IR), whereas KG-1 myeloblasts express negligible levels of IGF-IR (< 1% IGF-IR-positive cells). Exogenous IGF-I, IGF-II, and an IGF-I analog that binds poorly to IGF-binding protein-3 (des-IGF-I) increased DNA synthesis of HL-60 and U-937 cells in a dose-dependent (1-25 ng/ml) fashion by 2- to 4-fold in serum-free medium, whereas KG-1 cells did not respond to any of these growth factors. The IGF-induced increase in proliferation of HL-60 promyeloid cells was inhibited by soluble IGF-binding protein-3 (500 ng/ml) when these cells were stimulated with 10 ng/ml of either IGF-I (53 +/- 8%) or IGF-II (59 +/- 8%), but not with des-IGF-I (3 +/- 1%). In contrast, the anti-IGF-IR monoclonal antibody (mAb; alpha IR-3) inhibited the DNA synthesis caused by 10 ng/ml exogenous IGF-I (67 +/- 6%), IGF-II (72 +/- 8%), and des-IGF-1 (82 +/- 9%). Proliferation of KG-1 myeloblasts, however, was neither stimulated by the IGFs nor inhibited by the anti-IGF-IR mAb. In the absence of exogenous IGF-I, the mAb directed against the IGF-IR significantly suppressed basal DNA synthesis of HL-60 promyeloid (72 +/- 5%) and U-937 monocytic (39 +/- 7%) cells, but did not affect DNA synthesis of KG-1 myeloblasts (8 +/- 1%) compared to an isotype-matched control mAb. Similarly, the alpha IR-3 mAb abrogated vitamin D3-induced differentiation of the HL-60 cells into macrophages in serum-free medium, as assessed by expression of the leucam surface protein, CD11b. As the alpha IR-3 mAb inhibits DNA synthesis in the presence and absence of exogenous IGF-I on receptor-bearing cells, but not IGF-IR-negative cells, these data demonstrate that both endocrine and autocrine IGF-I are potent growth factors in human myeloid cells where expression of the surface receptor, rather than the ligand, is the critical control element. More importantly, these data support the hypothesis that autocrine IGF-I may play a significant role in the differentiation of promyeloid cells into macrophages.

Bone Marrow Cells↗
PubMed1996

Relationship between cytotoxicity and site-specific DNA recombination after in vitro exposure of leukemia cells to etoposide.

BACKGROUND: Etoposide, an inhibitor of the normal religation activity of the nuclear enzyme topoisomerase II, can induce a secondary acute myeloid leukemia characterized by site-specific DNA rearrangements. The schedule of drug administration appears to be a clinical risk factor for this devastating treatment complication. PURPOSE: We tested the hypothesis that prolonged exposure of leukemia cells in vitro to low concentrations of etoposide, compared with short exposures to high concentrations, could produce equivalent or greater desired cytotoxic effects, with decreased occurrence of undesired site-specific double-stranded DNA recombinational events (i.e., recombinogenesis). METHODS: We used the frequency of V(D)J (variable-diversity-joining) recombinase-mediated deletions of exons 2 and 3 of the hypoxanthine phosphoribosyltransferase (HPRT) gene as a biomarker of etoposide-induced, nonhomologous, site-specific DNA rearrangement. A polymerase chain reaction-based technique was used to measure exon 2 + 3 deletions in human lymphoid leukemia CCRF-CEM cells 6 days after either 4-hour or 24-hour treatment with etoposide at clinically relevant concentrations. Cytotoxic effects of etoposide (determined by the number of viable cells present in the treated compared with the control [i.e., untreated] cells) were measured 6 days after treatment of the cells. The frequency of the exon 2 + 3 deletion following the two treatment-duration conditions was compared by use of the Mantel-Haenszel statistic. All P values resulted from two-sided tests. RESULTS: Cytotoxicity increased with increasing etoposide concentration and exposure duration, as expected. By day 6, the frequency of exon 2 + 3 deletions was significantly higher (global P value = .0003) after the 4-hour treatment than after the 24-hour treatment, regardless of whether the frequency was assessed at etoposide concentrations achieving equivalent (e.g., 95%) cytotoxicity (14.2 x 10(-7) versus 4.1 x 10(-7) or at equivalent etoposide concentrations (e.g., 1 microM) (10.8 x 10(-7) versus 1.3 x 10(-7). Thus, the ratio of desired cytotoxic to undesired recombinogenic effects was higher with the 24-hour schedule. After the treated cells were subcloned at limiting dilutions, the frequency of the exon 2 + 3 deletion increased from 16.3 x 10(-7) to 4.33 x 10(-3), indicating that the recombinational event is not necessarily lethal. CONCLUSION: For all drug concentrations and levels of cytotoxicity studied in CCRF-CEM cells, there was a greater ratio of cytotoxicity to genetic recombination following prolonged exposure to etoposide than following brief exposure. IMPLICATION: These data suggest that recombinogenesis is not inextricably linked to cytotoxicity. If confirmed in the clinical setting, the use of prolonged dosage schedules may provide a means to decrease the risk of etoposide-induced acute myeloid leukemia without compromising treatment efficacy.

Acute Disease↗
PubMed1996

Overt testicular disease at diagnosis is associated with high risk features and a poor prognosis in patients with childhood acute lymphoblastic leukemia.

BACKGROUND: The impact of overt testicular disease on survival was assessed among patients treated at the study institution for childhood acute lymphoblastic leukemia (ALL) over a 15-year period. To the authors' knowledge, the frequency of overt testicular involvement at diagnosis of ALL and its impact on treatment outcome have not been reported previously. METHODS: The medical records of all 651 boys with ALL enrolled on St. Jude Total Therapy Studies X-XIIIA (May 1979 to December 1993) were reviewed to determine the frequency of overt testicular involvement at diagnosis. The log rank test and sequential Cox regression analyses, adjusted for known adverse features, were used to compare event free and overall survival for patients with and without testicular leukemia. A matched-pairs analysis was then conducted for both outcome measures. RESULTS: Thirteen of the 651 male patients (1.9%) presented with overt testicular leukemia. Compared with the other patients, these 13 boys had a significantly higher frequency of infant or adolescent age at diagnosis, hyperleukocytosis, splenomegaly, and mediastinal mass; a poorer 5-year event free survival (38% [95% confidence interval (CI), 15.4-61.4%] vs. 58% [95% CI, 53.6-61.7%], P = 0.06, log rank test); and a significantly poorer 5-year overall survival (37% [95% CI, 14-60.4%] vs. 75% [95% CI, 71.3-78.4%], P = 0.002). The difference in overall, but not event free, survival was supported by sequential Cox regression analyses and by the matched-pairs analysis (P = 0.04). CONCLUSIONS: Overt testicular involvement with leukemia at diagnosis was associated with an adverse survival in boys with ALL. Testicular irradiation may not be necessary in those patients who present with overt testicular involvement. This finding merits prospective evaluation in a larger sample of similarly treated patients.

Adolescent↗
PubMed1996

N-Ethylmaleimide inactivates a nucleotide-free Hsp70 molecular chaperone.

Hsp70 molecular chaperones are ATPases that bind to hydrophobic regions of proteins and guide their folding, assembly, and translocation across membranes. The ability of purified Hsp70s to uncoat clathrin-coated vesicles or to stimulate the post-translational translocation of precursor proteins into the endoplasmic reticulum, mitochondria, and the nucleus was previously shown not to be sensitive to the sulfhydryl-modifying reagent N-ethylmaleimide (NEM). During purification of factors required for protein folding in the cytosol, we found that the ATP-agarose binding activity of the yeast Hsp70 Ssa1p in postribosomal supernatants was inhibited by NEM. We also found that completely removing nucleotides from purified Ssa1p rendered its ATP-agarose binding activity, ATPase activity, and post-translational translocation-stimulating activity sensitive to NEM. We modified nucleotide-free Ssa1p with [14C]NEM and then digested it with proteases. Purification and sequencing of the radiolabeled proteolytic fragments revealed that each of Ssa1p's three cysteine residues (Cys-15, Cys-264, and Cys-303) was modified with [14C]NEM. ADP protected each of the cysteine residues from modification and protected Ssa1p from inactivation. The cysteine residues are the reactive centers of three NEM-reactive sites (NRS1-3). A comparison of Ssa1p's NRSs to sequences of other Hsp70s and actin revealed that Cys-15 of NRS1 is highly conserved and that sensitivity to NEM may be a property of many Hsp70s. Based on the three-dimensional structure of Hsc70, the predicted locations of Ssa1p's cysteine residues suggest that NEM may disrupt the conformation of Ssa1p or interfere with its ability to bind nucleotides. Together the results demonstrate that Ssa1p is an NEM-sensitive factor in cytosolic extracts from yeast that stimulates post-translational translocation of proteins into organelles.

Adenosine Triphosphatases↗
PubMed1996

A murine host cell factor required for nicking of the dimer bridge of MVM recognizes two CG nucleotides displaced by 10 basepairs.

During replication of the minute virus of mice (MVM) genome, a dimer replicative form (RF) intermediate is resolved into two monomer RF molecules in such a way as to retain a unique sequence within the left hand hairpin terminus of the viral genome. Although the proposed mechanism for resolution of the dimer RF remains uncertain, it likely involves site-specific nicking of the dimer bridge. The RF contains two double-stranded copies of the viral genome joined by the extended 3' hairpin. Minor sequence asymmetries within the 3' hairpin allow the two halves of the dimer bridge to be distinguished. The A half contains the sequence [sequence: see text], whereas the B half contains the sequence [sequence: see text]. Using an in vitro assay, we show that only the B half of the MVM dimer bridge is nicked site-specifically when incubated with crude NS-1 protein (expressed in insect cells) and mouse LA9 cellular extract. When highly purified NS-1, the major nonstructural protein of MVM, is used in this nicking reaction, there is an absolute requirement for the LA9 cellular extract, suggesting a cellular factor (or factors) is (are) required. A series of mutations were created in the putative host factor binding region (HFBR) on the B half of the MVM dimer bridge adjacent to the NS-1 binding site. Nicking assays of these B half mutants showed that two CG motifs displaced by 10 nucleotides are important for nicking. Gel mobility shift assays demonstrated that a host factor(s) can bind to the HFBR of the B half of the dimer bridge and efficient binding depends on the presence of both CG motifs. Competitor DNA containing the wild-type HFBR sequence is able to specifically inhibit nicking of the B half, indicating that the host factor(s) bound to the HFBR is(are) essential for site-specific nicking to occur.

Animals↗
PubMed1996

Afferent connections to the dorsal nucleus of the lateral lemniscus of the mustache bat: evidence for two functional subdivisions.

The dorsal nucleus of the lateral lemniscus (DNLL) of the mustache bat, Pteronotus parnellii, was found to consist of two divisions. The neurons in each division were distinguished by their temporal discharge patterns evoked both by tone bursts and sinusoidal amplitude-modulated (SAM) signals. Neurons in the anterior one-third of the DNLL responded to tone bursts with an onset discharge pattern and only phase-locked to SAM signals with low modulation frequencies (< 300 Hz). Neurons in the posterior two-thirds of the DNLL responded to tone bursts with a sustained discharge pattern and phase-locked to SAM signals with much higher modulation frequencies (400-800 Hz). In addition, there was a different frequency representation in the two divisions. The frequency representation in the posterior division was from about 15 to 120 kHz, whereas in the anterior division it was only up to 62 kHz. The physiological differences were further supported by data from experiments that revealed the sources of afferent projections to the two DNLL divisions. Retrograde labeling showed that the afferent projections to the two divisions were from different neuronal populations. Input differences were of two types. Some nuclei projected to one or the other DNLL division, but not to both. For instance, the ventral nucleus of the lateral lemniscus projected predominately to the anterior DNLL and provided little or no inputs to the posterior DNLL, whereas the medial superior olive innervated the posterior but not the anterior DNLL. Other lower nuclei projected to both DNLL divisions. These include the contralateral cochlear nucleus, the ipsi- and contralateral lateral superior olives, the intermediate nucleus of the lateral lemniscus, and the contralateral DNLL. However, the projections to each division of the DNLL originate from different neuronal subpopulations in each lower nucleus. The functional implications of these findings are discussed in the context of the possible impacts that the two DNLL divisions exert on their postsynaptic targets in the inferior colliculus.

Acoustic Stimulation↗
PubMed1996

Etoposide causes illegitimate V(D)J recombination in human lymphoid leukemic cells.

Etoposide is one of the most widely used antineoplastics. Unfortunately, the same treatment schedules associated with impressive efficacy are associated with an increased risk of secondary acute myeloid leukemia (AML), which has prompted its withdrawal from some treatment regimens, thereby potentially compromising efficacy against the original tumor. Because etoposide-associated AML is characterized by site-specific illegitimate DNA recombination, we studied whether etoposide could directly cause site-specific deletions of exons 2 and 3 in the hprt gene. Human lymphoid CCRF-CEM cells were treated with etoposide for 4 hours, and DNA was isolated after subculturing. The deletion of exons 2 and 3 from hprt was assayed by a quantitative polymerase chain reaction (PCR) method. In the absence of etoposide treatment, the frequency of deletions of exons 2 and 3 was very low (5.05 x 10(-8)). After exposure to 10 mumol/ L etoposide, the frequency of the exon 2 + 3 deletion was increased immediately after and at 24 hours after etoposide treatment (65 to 89 x 10(-8)) and increased to higher levels (128 to 173 x 10(-8)) after 2 and 6 days of subculture (P < .001 overall). The frequency of the exon 2 + 3 deletion assessed at 6 days of subculture after 4 hours of 0, 0.25, 1, 2.5, 5, and 10 mumol/L etoposide treatment increased with etoposide concentration, ie, 5.05 x 10(-8), 89.2 x 10(-8), 108 x 10(-8), 142 x 10(-8), 163 x 10(-8), and 173 x 10(-8), respectively (P < .0001). Sequencing of a subset of amplified products confirmed the presence of DNA sequences at the breakpoints consistent with V(D)J recombination. By contrast, exon 2 + 3 deletions after etoposide treatment in the myeloid cell lines KG-1A and K562 showed no evidence of V(D)J recombinase in their genesis. We conclude that etoposide can induce the illegitimate site-specific action of V(D)J recombinase on an unnatural DNA substrate after a single treatment in human lymphoid cells.

Base Sequence↗
PubMed1996

Cloning of the human sodium lodide symporter.

The iodide concentrating activity of the thyroid gland is essential to the production of thyroid hormone and also provides a mechanism for the treatment of thyroid cancer by radioiodine ablation. We report here the nucleotide and amino acid sequence of the human sodium iodide symporter (hNIS), which mediates the iodide uptake activity in the thyroid gland. An open reading frame of 1929 nucleotides encodes a protein of 643 amino acids with 84% identity to the rat NIS (rNIS). Transient expression of the hNIS cDNA conferred perchlorate-sensitive iodide uptake to a nonthyroid cell line, COS-7. The expression of hNIS was detected at variable levels in papillary thyroid carcinoma tissues but not in any of the thyroid carcinoma cell lines that have lost the iodide uptake activity.

Amino Acid Sequence↗
PubMed1996

Site-directed mutagenesis of the 100-kDa subunit (Vph1p) of the yeast vacuolar (H+)-ATPase.

Vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for acidification of intracellular compartments in eukaryotic cells. V-ATPases possess a subunit of approximate molecular mass 100 kDa of unknown function that is composed of an amino-terminal hydrophilic domain and a carboxyl-terminal hydrophobic domain. To test whether the 100-kDa subunit plays a role in proton transport, site-directed mutagenesis of the VPH1 gene, which is one of two genes that encodes this subunit in yeast, has been carried out in a strain lacking both endogenous genes. Ten charged and twelve polar residues located in the seven putative transmembrane helices in the COOH-terminal domain of the molecule were individually changed, and the effects on proton transport, ATPase activity, and assembly of the yeast V-ATPase were measured. Two mutations (R735L and Q634L) in transmembrane helix 6 and at the border of transmembrane helix 5, respectively, showed greatly reduced levels of the 100-kDa subunit in the vacuolar membrane, suggesting that these mutations affected stability of the 100-kDa subunit. Two mutations, D425N and K538A, in transmembrane helix 1 and at the border of transmembrane helix 3, respectively, showed reduced assembly of the V-ATPase, with the D425N mutation also reducing the activity of V-ATPase complexes that did assemble. Two mutations, H743A and K593A, in transmembrane helix 6 and at the border of transmembrane helix 4, respectively, have significantly greater effects on activity than on assembly, with proton transport and ATPase activity inhibited 40-60%. One mutation, E789Q, in transmembrane helix 7, virtually completely abolished proton transport and ATPase activity while having no effect on assembly. These results suggest that the 100-kDa subunit may be required for activity as well as assembly of the V-ATPase complex and that several charged residues in the last four putative transmembrane helices of this subunit may play a role in proton transport.

Adenosine Triphosphate↗
PubMed1996

Effectiveness of intensified rotational combination chemotherapy for late hematologic relapse of childhood acute lymphoblastic leukemia.

Relapsed acute lymphoblastic leukemia (ALL) usually carries a dire prognosis. We evaluated the effectiveness and long-term complications of intensive rotational combination chemotherapy for late hematologic relapse (median, 16 months after elective cessation of therapy) among 34 children and young adults (ages 4 to 23 years). Concurrent central nervous system (CNS) relapse was present in 3 cases and testicular relapse in 4. Secondary therapy comprised an intensive five-drug reinduction (6 weeks) followed by continuation treatment with four drug pairs, rotated weekly in 4-week cycles over 120 weeks. Intrathecal chemotherapy (methotrexate, hydrocortisone, cytarabine) was given three times during reinduction and every 8 weeks during continuation. Treatment was electively discontinued at week 120 in the absence of detectable disease. Thirty-three patients (97%) attained a second complete remission. At a median follow-up of 9.3 years (range, 4.5 to 11.4), estimates of 5-year second event-free and overall survival (+/- SE) are 65% +/- 8% and 79% +/- 7%, respectively. Eleven patients had a second relapse (9 marrow, 2 testicular) and one developed secondary myeloid leukemia. There have been no CNS relapses or deaths in remission. Treatment was well-tolerated and was given largely on an outpatient basis. Late effects are primarily endocrinologic; one child had a second malignant solid tumor (presumed related to initial radiation therapy) that was treated successfully. Intensive treatment with alternating non-cross-resistant drug pairs for late hematologic relapses of ALL is effective and well-tolerated, and produces results similar to those achieved in patients with newly diagnosed ALL. Event-free survival compares favorably with reports of other relapse regimens, including those incorporating bone marrow transplantation.

Adolescent↗
PubMed1996

A novel nuclear structure containing the survival of motor neurons protein.

Spinal muscular atrophy (SMA) is a common, often fatal, autosomal recessive disease leading to progressive muscle wasting and paralysis as a result of degeneration of anterior horn cells of the spinal cord. A gene termed survival of motor neurons (SMN), at 5q13, has been identified as the determining gene of SMA (Lefebvre et al., 1995). The SMN gene is deleted in > 98% of SMA patients, but the function of the SMN protein is unknown. In searching for hnRNP-interacting proteins we found that SMN interacts with the RGG box region of hnRNP U, with itself, with fibrillarin and with several novel proteins. We have produced monoclonal antibodies to the SMN protein, and we report here on its striking cellular localization pattern. Immunolocalization studies using SMN monoclonal antibodies show several intense dots in HeLa cell nuclei. These structures are similar in number (2-6) and size (0.1-1.0 micron) to coiled bodies, and frequently are found near or associated with coiled bodies. We term these prominent nuclear structures gems, for Gemini of coiled bodies.

3T3 Cells↗
PubMed1996

Rearrangement of the MLL gene confers a poor prognosis in childhood acute lymphoblastic leukemia, regardless of presenting age.

MLL gene rearrangements are associated with an extremely poor prognosis in infants with acute lymphoblastic leukemia (ALL), but little is known about their clinical significance in older children. Therefore, we studied 45 cases of childhood ALL with abnormalities of chromosome 11q23 for rearrangement of the MLL gene to determine if this feature confers a uniformly poor prognosis. MLL gene rearrangements were detected in all 18 cases with the common t(4;11), t(9;11) or t(11;19) translocations, whereas only 5 of 12 patients with either unbalanced or uncommon balanced translocations demonstrated a rearrangement. Abnormalities of the MLL gene were not detected in any of the 15 cases with a deletion or inversion of the chromosomes 11q23 region. The presence of an MLL rearrangement was significantly associated with age less than 1 year (P < .001), leukocyte count >50 x 10(9)/L (P = .003), and the absence of leukemic cell CD10 expression (P < .001). In a stratified statistical analysis adjusted for age and treatment protocol, MLL gene rearrangement was correlated with an inferior treatment outcome (P = .028). The 4-year event-free survival estimate (+/- SE) was 10% +/- 6.5% for cases with a rearranged MLL gene and 64% +/- 19.2% for other cases. When infants were excluded from the analysis, MLL rearrangement was still significantly associated with a poor outcome (P = .02), and remained so with the exclusion of t(4;11)-positive cases (P = .05). Thus, regardless of presenting age, MLL gene rearrangement identifies a high-risk subgroup of patients who are not likely to be cured with conventional treatment.

Age Factors↗
PubMed1996

Characterization of bovine endothelial nitric oxide synthase expressed in E. coli.

Bovine endothelial constitutive nitric oxide synthase (eNOS) was expressed in E. coli as a soluble, catalytically active enzyme using the pCW expression vector coexpressed with a plasmid, pGroELS, encoding the chaperonins groEL and groES. The E. coli BL21 cultures reproducibly synthesized 6-10 mg of recombinant enzyme per liter of culture. The eNOS protein was purified using 2'5'-ADP Sepharose 4B and appeared as a single band of apparent molecular mass 135 kDa on SDS/PAGE. The recombinant resting enzyme is predominantly high spin with an absorbance maximum at 406 nm. The dithionite-reduced, CO-bound form shows an absorbance maximum at 444 nm. The spectral properties of recombinant eNOS from E. coli are identical to those observed with eNOS from stably transfected HEK 293 cells or from baculovirus expression systems. Enzymatic activity of eNOS from E. coli ranged between 68-135 nmol product formed/min/mg at 25 degrees C, using hemoglobin-NO capture or L-citrulline formation assays. The enzyme is replete with heme and flavins and both activity and [3H]-nitroarginine binding were largely dependent on tetrahydrobiopterin. The heterologous expression of eNOS offers a number of advantages over tissue sources of the protein.

Animals↗