Sensitive measurement of quantity dynamics of FLT3 internal tandem duplication at early time points provides prognostic information.

BACKGROUND: The level of minimal residual disease (MRD) in acute myeloid leukemia (AML) at early time points (TPs) may be an important prognostic factor. Although internal tandem duplication of FLT3 (FLT3-ITD) as an MRD marker has been questioned for its instability based on semi-quantitative methods, we hypothesized that FLT3-ITD dynamics measured by sensitive quantitative real-time PCR at early TPs before appearance of instability may provide prognostic information. PATIENTS AND METHODS: We measured mutant quantity in 493 serial samples from 55 patients with a median follow-up time of 64.8 months. The FLT3-ITD quantities after induction (TP1) and after the first post-induction chemotherapy (TP2) were analyzed. RESULTS: We found that lower FLT3-ITD levels at TP2 predicted longer overall survival (OS) and disease-free survival (DFS) regardless of cytogenetic risk. Multivariate analysis showed that ≥3 log reduction of FLT3-ITD at TP2 independently predicted better DFS and a trend toward better OS. FLT3-ITD disappeared at relapse in 16.7% of patients and none in those harboring mutant NPM1 compared with 29.4% in those with wild-type NPM1 (P = 0.032). CONCLUSIONS: Though the mutation may disappear at relapse in a few patients, FLT3-ITD levels at early TPs after chemotherapy provide prognostic information. FLT3-ITD is significantly more stable in those with mutant NPM1.

Production of beta-galactosidase by Bifidobacteria as influenced by various culture conditions.

Beta-Galactosidase production by Bifidobacterium longum CCRC 15708, Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 was first examined with B. longum CCRC 15708 showing the highest production of beta-galactosidase and the highest specific activity. Further study with B. longum CCRC 15708 revealed that the highest level of beta-galactosidase was produced with lactose and yeast extract as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial pH of 6.5 and at 37 degrees C. Under these optimum culture conditions, a maximumbeta-galactosidase activity of 18.6 U/ml could be obtained after 16 h of fermentation in a medium contain 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4.7H2O and 0.03% L-cysteine. The highest transgalactosylation activity was also detected in this culture after 14-16 h of fermentation.

Identification of a unique binding protein specific for a novel retinoid inducing cellular apoptosis.

The retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN, CD437) induces apoptosis in a variety of cell types, many of which are cancer cells that resist the antiproliferative and/or differentiating effects of retinoids. While the retinoids exert their effects by binding to the retinoic acid nuclear receptors (RARs) or retinoid X receptors (RXRs), AHPN (CD437) binds to another protein with different ligand specificity. In nuclear extracts from HL-60R cells the binding of AHPN (CD437) was only minimally competed by either retinoic acid (tRA)or 9-cis-retinoic acid (9-cis-RA), the natural ligands for the RARs and RXRs, respectively. Moreover, AHPN (CD437) was unable to compete with either tRA or 9-cis-RA for binding to endogenous retinoid receptors in nuclear extracts from the MDA-MB-468 breast carcinoma cell line. Size exclusion chromatography revealed AHPN binding to a 95 kDa protein(s) which is neither an RAR or RXR. Our results suggest that apoptosis induction by AHPN (CD437) may occur through interaction with another protein and is independent of the RAR/RXR-signaling pathways.

Retinoid induced apoptosis in leukemia cells through a retinoic acid nuclear receptor-independent pathway.

Trans retinoic acid (RA) has proven to be a potent therapeutic agent in the treatment of acute promyelocytic leukemia. Unfortunately, other subtypes of acute myelogenous leukemia are resistant to the antiproliferative and differentiating effects of RA. In this report, we describe a novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN; CD437) that not only totally inhibits the proliferation of RA-resistant leukemic cell lines HL-60R and K562 but also induces apoptosis in these cells. Exposure of HL-60R to CD437 results in the rapid (within 30 minutes) increase of the cyclin-dependent kinase inhibitor p21(waf1/cip1) as well as GADD45 mRNA. Manifestations of CD437-mediated programmed cell death are noted within 2 hours, as indicated by both the cleavage and activation of the CPP32 protease and cleavage of poly (ADP-ribose) polymerase. This is followed by cleavage of bcl-2 and internucleosomal DNA degradation. HL-60R cells do not express the retinoid nuclear receptor RAR beta and RAR gamma and express a truncated RAR alpha. Thus, CD437 induction of p21(waf1/cip1) and GADD45 mRNAs and apoptosis occurs through a unique mechanism not involving the retinoid nuclear receptors. CD437 represents a unique retinoid with therapeutic potential in the treatment of myeloid leukemia.

The primary structure of a protein carboxyl methyltransferase from bovine brain that selectively methylates L-isoaspartyl sites.

Protein L-isoaspartyl methyltransferase (PIMT) transfers the methyl group of S-adenosyl-L-methionine to free alpha-carboxyl groups of atypical L-isoaspartyl residues in proteins. The complete primary structure of the type I isoform of bovine brain PIMT was determined by sequence analysis of peptides generated by endoprotease Lys-C, trypsin, cyanogen bromide, and endoprotease Asp-N digests. The correct composition of every peptide was verified by fast atom bombardment mass spectrometry. The efficiency of sequencing by tandem mass spectrometry was examined for several peptides by comparing its speed and accuracy with automated Edman degradation. Tandem mass spectrometry was used to determine the structure of the NH2-terminal blocked peptide derived from a hydroxylamine cleavage. PIMT is 226 residues with Mr = 24,500 and contains acetyl alanine as the amino-terminal residue. The partial sequence (141 residues from 8 tryptic peptides) of a homologous human red cell PIMT (Gilbert, J. M., Fowler, A., Bleibaum, J., and Clarke, S. (1988) Biochemistry 27, 5227-5233) shows a 97% identity with the corresponding peptides of the bovine brain enzyme. The complete brain enzyme sequence reported here bears no significant homology to any other known class of methyltransferase including those which methylate the side chain gamma-carboxyl group of receptor proteins involved in bacterial chemotaxis.

A case of malignant lymphoma, undifferentiated, Burkitt's type with high antibodies to Epstein-Barr virus.

A 5-year-4-month-old girl was admitted to Taipei Municipal Jen-Ai Hospital because of a rapidly enlarging right submandibular tumor. The diagnosis of malignant lymphoma, undifferentiated, Burkitt's type was made by distinctive histopathological features. Positive serological findings related to Epstein-Barr virus infection were disclosed. Complete remission has been achieved since the treatment with high-dose cyclophosphamide was given, and no signs of relapse have been found yet.