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.

Cultural condition affecting the growth and production of beta-galactosidase by Bifidobacterium longum CCRC 15708 in a jar fermenter.

In the present study, the growth and production of beta-galactosidase by Bifidobacterium longum CCRC 15708 in a 5-L jar fermenter as influenced by cultivation temperature (27-42 degrees C), medium pH (4.5-7.5) and agitation speed (5-200 rpm) were evaluated. In general, it was found that a cultivation temperature of 37 degrees C proved optimal for both growth and beta-galactosidase production by the test organism. Although the growth of the test organism was the highest in the culture with pH controlled at 4.5-6.5, the culture with pH controlled at 6.5 resulted in the highest production of beta-galactosidase. Further, agitation at 100 rpm or more was found to enhance both the growth and production of beta-galactosidase. Fermentation conducted in a jar fermenter having the pH of the culture medium, the cultivation temperature, and the agitation speed controlled at 6.5, 37 degrees C, and 100 rpm, respectively, a maximum beta-galactosidase activity of 36.7 U/ml and a maximum transgalactosylation activity of 0.49 U/ml was achieved in 10 h of fermentation. There are ca 2.0 and 12.3 fold greater than the reported maximum beta-galactosidase and transgalactosylation activity, respectively, produced by B. longum CCRC 15708 in a flask culture system.

Enzymatic production of galactooligosaccharides by beta-galactosidase from Bifidobacterium longum BCRC 15708.

The production of galactooligosaccharides (GOSs) by transgalactosylation using beta-galactosidase from Bifidobacterium longum BCRC 15708 was studied. Other than lactose, galactose, and glucose, two types of GOSs, tri- and tetrasaccharides, were formed after beta-galactosidase action on 40% lactose. Trisaccharides were the major type of GOS formed. Generally, an increase of the initial lactose concentration in the reaction mixture resulted in a higher GOS production. A maximum yield of 32.5% (w/w) GOSs could be achieved from 40% lactose solution at 45 degrees C, pH 6.8, when the lactose conversion was 59.4%. The corresponding productivity of GOSs was 13.0 g/(L.h). Transgalactosylation activity of beta-galactosidase from a test organism showed a relatively lower sensitivity toward glucose and galactose than that from other organisms. The addition of 5% or 10% glucose or galactose to the reaction mixture did not significantly (p>0.05) reduce the transgalactosylation reaction of beta-galactosidase.

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.

Anti-lewisite activity and stability of meso-dimercaptosuccinic acid and 2,3-dimercapto-1-propanesulfonic acid.

Meso-dimercaptosuccinic acid (DMSA) and the sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS) are analogous in chemical structure to dimercaprol (BAL, British Anti-Lewisite). Dimercaprol was among the first therapeutically useful metal chelating agents and was developed originally as an anti-lewisite agent. Either DMSA or DMPS protects rabbits from the lethal systemic action of dichloro(2-chlorovinyl)arsine (29.7 mumols/kg, also known as lewisite. The analogs are active in this respect when given either sc or po. The stability of each of the three dimercapto compounds in distilled H2O, pH 7.0 at 24 degrees, has been examined for seven days. DMSA retained 82% of its mercapto groups, but no titratable mercapto groups remained in the DMPS or BAL solutions. At pH 5.0, however, there was no striking difference in the stability of the three dimercapto compounds (78-87%) over a seven day period. DMSA and DMPS warrant further investigation as water soluble metal binding agents in both in vivo and in vitro experiments.

Thymine 7-hydroxylase and pyrimidine deoxyribonucleoside 2' -hydroxylase activities in Rhodotorula glutinis.

Cell-free preparations from Rhodotorula glutinis catalyzed the conversion of deoxyribonucleosides to ribonucleosides in a pyrimidine deoxyribonucleoside 2' -hydroxylase reaction. The reaction occurred with only thymidine or deoxyuridine, of the common deoxyribonucleosides, without detachment of the deoxyribose moiety, at the nucleoside level. The same enzyme preparations catalyzed the conversion of thymine to 5-hydroxymethyluracil in a thymine 7-hydroxylase reaction. Requirements for molecular oxygen, alpha-ketoglutarate, Fe2+, and ascorbate indicated that the 2' -hydroxylase and 7-hydroxylase reactions are of the alpha-keto-acid dioxygenases class. The requirements for alpha-ketoglutarate and Fe2+ were very stringent. During the course of the 2' -hydroxylase and 7-hydroxylase reactions, alpha-ketoglutarate was decarboxylated to form succinate and CO2 so that the ratio of hydroxylated nucleoside or pyrimidine to CO2 was 1:1.5-Hydroxymethyluracil and 5-formyluracil also stimulated the decarboxylation of alpha-ketoglutarate and thus appeared to undergo 7-hydroxylase reactions.

Metabolism of pyrimidine deoxyribonucleosides in Neurospora crassa.

The experiments in this report involve the following series of reactions which were previously demonstrated with purified enzyme preparations from Neurospora crassa: thymidine a yields thymine ribonucleoside b yields thymine c yields 5-hydroxymethyluracil d yields 5-formyluracil e yields uracil-5-carboxylic acid f yields uracil. The evidence for some of the reactions occurring in vivo has been incomplete and for others totally lacking. In this paper intact cells of Neurospora are shown to be capable of converting the substrates of each of the reactions to the corresponding products. Studies are described which were carried out in vivo and in vitro with the pyrimidineless strains pyr-4,uc-1,uc-2 and pyr-4,uc-1,uc-3, developed by Williams and Mitchell. The results reported in the present paper indicate that (reaction a) and the uc-3 mutation affects thymine 7-hydroxylase (reactions c,d, and e). Evidence is presented for the 2'-hydroxylase reaction being the major, if not only, way by which Neurospora can initiate the conversion of thymidine to the pyrimidines of nucleic acids and for the 2'-hydroxylation of thymidine and deoxyuridine being catalyzed by the same enzyme. Deoxycytidine was shown not to be hydroxylated in intact cells but instead deaminated to deoxyuridine, which in turn was converted to uridine. Further studies with the uc-3-carrying strain showed that an enzyme other than thymine 7-hydroxylase can also convert 5-formyluracil to uracil-5-carboxylic acid.

DL- and meso-dimercaptosuccinic acid: in vitro and in vivo studies with sodium arsenite.

Dimercaptosuccinic acid (DMSA) has been receiving increasing interest as an antidote for poisoning by heavy metals. Of the various isomeric forms, meso-DMSA has been studied most extensively because its preparation and purification are relatively easy. Using a variety of in vitro and in vivo procedures, we have investigated and compared DL- and meso-DMSA as antidotes for sodium arsenite. The two forms of DMSA are equally effective in preventing or reversing, in vitro, the arsenite inhibition of the activity of mouse kidney pyruvate dehydrogenase (PDH) complex, DL-DMSA, however, is superior to meso-DMSA for the in vivo reversal of PDH activity as measured in vitro. The LD50 values of DL- and meso-DMSA in the mouse were 10.84 and 13.73 mmol/kg, ip, respectively. The ED50 values of the two DMSA forms were not significantly different in mice receiving a LD99 dose of sodium arsenite. DL- and meso-DMSA were equally effective in mobilizing tissue 74As of rabbits. The activity of DMSA as an arsenic antidote appeared to be independent of its isomeric structural configuration. There did not appear to be any great advantage in using DL-DMSA instead of meso-DMSA as an arsenic antidote.

Optical isomers of 2,3-dimercapto-1-propanesulfonate: antidotal activity, in vitro and in vivo, against sodium arsenite.

DMPS (2,3-dimercapto-1-propanesulfonate, Na salt) is an important water soluble analog of dimercaprol. All investigations of this antidote for heavy metal intoxication have dealt only with the racemic mixture. In the present report, the optical isomers of DMPS have been separated and the arsenic-antidote activity of the levo-rotatory (-)-isomer, the dextro-rotatory (+)-isomer and the racemic mixture of DMPS have been investigated in vivo and in vitro. The individual optical isomers and the racemic mixture of DMPS are effective equally, in vitro, in preventing the inhibition by sodium arsenite of the activity of mouse kidney pyruvate dehydrogenase complex. In addition, when pyruvate dehydrogenase is inhibited, in vitro, by sodium arsenite, any of the three DMPS preparations will reverse the inhibition equally well. The in vitro evidence suggests that two molecules of DMPS are required to prevent the effects of one molecule of sodium arsenite. Neither the LD50 nor the ED50 values of each of the three forms of DMPS differ significantly when measured i.p. in mice. In addition, there is no striking difference between the effectiveness of the levo- or dextro-rotatory DMPS when given orally to mice challenged with sodium arsenite. Thus, the use of the individual optical isomers of DMPS does not appear to have any advantage over the racemic mixture as an arsenic antidote under these conditions.

Uracil's uncoupling of the decarboxylation of alpha-ketoglutarate in the thymine 7-hydroxylase reaction of Neurospora crassa.

Highly purified preparations of thymine 7-hydroxylase from Neurospora crassa catalyzed the decarboxylation of alpha-ketoglutarate but yielded no hydroxylated product when uracil was substituted for thymine in the standard incubation mixture. Although the uracil-dependent decarboxylation was much slower than the coupled reaction, both reactions were similar with respect to the requirement for molecular oxygen, the stoichiometric formation of succinate, and the stimulations effected by Fe2+, ascorbate, and catalase. That the same enzyme catalyzed both reactions was indicated by the parallel loss of the uracil- and thymine-dependent activities upon heat denaturation, their copurification, and the lower level of both activities in a mutant strain deficient in the 7-hydroxylase. These data are consonant with molecular oxygen initially attacking alpha-ketoglutarate in the thymine 7-hydroxylase reaction.

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.

Effects of natriuretic peptides on vascular smooth-muscle cells derived from different vascular beds.

1. This study explored the hypothesis that atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-natriuretic peptide (CNP) have differing antiproliferative and antihypertrophic effects on pulmonary artery (PA) and thoracic aorta (TA) smooth-muscle cells (SMCs). 2. Cultured cells were exposed to 5% fetal calf serum (FCS) and angiotensin II (A-II) to induce DNA and protein synthesis, respectively. 3. ANP (10(-7) M) significantly reduced thymidine uptake in TA by 31% +/- 2% (P < or = 0.01) but not in PA (P > or = 0.05). 4. In parallel experiments, BNP (10(-7) M) significantly reduced thymidine uptake in TA (-22% +/- 5%, P < or = 0.01), but not in PA cells (P > or = 0.05). 5. CNP (10(-7) M) did not significantly alter thymidine uptake in TA cells exposed to FCS, but it did significantly reduce uptake in PA (-28.5% +/- 4%) 2(P < or = 0.05). 6. Blunting by ANP (10(-7) M) of the A-II (10(-8) M)-induced increase in protein synthesis was significantly greater in PA than in TA cells. 7. However, BNP and CNP (10(-7) M) exerted similar antihypertrophic effects on TA and PA cells exposed to A-II. 8. The antiproliferative effects of BNP and ANP exceed those of CNP in TA SMCs, but CNP appears to be the most effective antiproliferative agent in PA SMCs. In addition, PA-derived SMCs are more sensitive to the antihypertrophic effects of ANP than TA-derived cells, suggesting phenotypic differences. The findings indicate that the natriuretic peptides may play complementary roles in modulating SMC proliferation and protein synthesis.

DMSA, DMPS, and DMPA--as arsenic antidotes.

meso-Dimercaptosuccinic acid (DMSA), 2,3-dimercapto-1-propanesulfonic acid, Na salt (DMPS), and N-(2,3- dimercaptopropyl )- phthalamidic acid (DMPA) are water soluble analogs of 2,3-dimercapto-1-propanol (BAL). The relative effectiveness or therapeutic index of these dimercapto compounds in protecting mice from the lethal effects of an LD99 of sodium arsenite is DMSA greater than DMPS greater than DMPA greater than BAL in the magnitude of 42:14:4:1, respectively. DMPS, DMPA, or DMSA will mobilize tissue arsenic. BAL, however, increases the arsenic content of the brain of rabbits injected with sodium arsenite. These results raise the question as to the appropriateness of BAL as the treatment for systemic arsenic poisoning. Either DMSA or DMPS, when given sc or po, will protect rabbits against the lethal systemic effects of subcutaneously administered Lewisite . DMPS and DMSA have promise as prophylactics for the prevention of the vesicant action of Lewisite . The sodium arsenite inhibition of the pyruvate dehydrogenase (PDH) complex can be prevented and reversed in vitro or in vivo by DMPS, DMSA, DMPA, or BAL. Of them all, DMPS is most potent and BAL appears to be the least potent. The usefulness of all these dimercapto compounds would be enhanced by a careful study of their metabolism and biotransformation. These dimercapto compounds are in a great many respects orphan drugs. At this stage of their development, it is very difficult for the clinician to obtain funds to study them clinically even though they appear to be useful for treatment of poisoning by any one of the heavy metals.

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.

N-(2,3-dimercaptopropyl)phthalamidic acid: protection, in vivo and in vitro, against arsenic intoxication.

The ip LD50s of N-(2,3-dimercaptopropyl)phthalamidic acid (DMPA) and British Anti-Lewisite (BAL) were 0.819 and 1.48 mmol/kg, respectively, in male albino mice. The ip ED50 of DMPA and BAL for prevention of the lethal effects of 0.15 mmol NaAsO2/kg was 0.022 and 0.169 mmol/kg, respectively. DMPA increased the LD50 of sodium arsenite by approximately 2.5-fold following two ip injections of 0.20 mmol DMPA/kg. The effectiveness of DMPA in reducing the toxicity of NaAsO2 was further demonstrated by its reversal of the sodium arsenite inhibition of pyruvate dehydrogenase multienzyme complex (PDH) activity in vitro. Similarly, in an in vivo experiment in which mice received 0.10 mmol NaAsO2/kg, and 30 min later were given 0.05 or 0.10 mmol/kg DMPA, there was a rapid recovery of PDH activity. The distribution of 74As in the tissues of male New Zealand rabbits was altered following im injection of 0.20 mmol/kg DMPA. Under these conditions, the tissue concentration of 74As was significantly decreased. For all tissues tested, the 74As content decreased by at least 50% as compared to that of untreated controls. DMPA was effective also in increasing both urinary and fecal excretion of arsenic. The stability of aqueous solutions of DMPA varies with the pH of the solution. DMPA is more stable in acid solution.

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.

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.