Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14⁺ and CD33⁺ cells but to a reduction in CD34⁺ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14⁺ and CD33⁺ as well as CD34⁺ cells but in a significant increase in CD3⁺ and CD56⁺ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

Purification of acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase from Catharanthus roseus leaves

The enzyme acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase which terminates vindoline biosynthesis has been isolated from Catharanthus roseus leaves, further characterized and purified to homogeneity by three step column chromatography and subsequent preparative isoelectric focusing. Kinetic properties concerning the enzyme reaction are discussed. Five multiple forms of the acetyl-transferase could be observed, each consisting of two subunits. This enzyme is now the best characterized of the enzymes involved in vindoline biosynthesis.Abbreviations DTE dithiothreitol - EDTA ethylenediamine-tetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid - IEF isoelectric focusing - KP_i potassium phosphate - M_r rel.molecular mass - PEG polyethylene glycol - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Effects of the 5-lipoxygenase inhibitors AA-863 and U-60,257 on human glioma cell lines

The effects of two specific 5-lipoxygenase inhibitors AA-863 and U-60,257 (piriprost) on the growth of two human glioma cell lines, U-343 MGa and U-251 MG were investigated. Both monolayer cultured cells and spheroids were studied. The results of the monolayer studies showed potent and dose dependent inhibitory effects on the proliferation of glioma cells (IC50/one week treatment/of AA-863: 9.0 microM, IC50 of U-60,257: 40.0 microM). The experiments made on the tumor spheroids suggested an inhibitory effect on proliferation and volume growth already at lower doses (AA-863: 0.4-2.0 microM, U-60,257: 1.0-5.0 microM), a dose range where effects were not found in monolayers. At higher doses (AA-863: 10.0-30.0 microM, U-60,257: 30.0-90.0 microM) the experiments with spheroids failed to demonstrate a further inhibitory effect on spheroid volume, probably attributed to phenomena such as swelling of cells, dissociation of spheroid structure and development of necrosis. The clearly dose dependent inhibitory effect on the proliferation of human glioma cells in monolayer culture and the inhibitory effects on spheroid growth with these specific inhibitors indicate a role for lipoxygenase products in the growth of gliomas.     

Six year follow up of infants with bacteriuria on screening.

OBJECTIVE--To determine the value of screening for bacteriuria in infants with special emphasis on the natural course of untreated asymptomatic bacteriuria, renal growth, and renal damage. DESIGN--Prospective six year follow up of infants with bacteriuria on screening in an unselected infant population. SETTING--Paediatric outpatient clinic. PATIENTS--50 Infants (14 girls, 36 boys) with bacteriuria on screening verified by suprapubic aspiration from an unselected population of 3581 infants in a defined area of Gothenburg. INTERVENTIONS--Children with asymptomatic bacteriuria and normal findings on initial urography were untreated, although other infections were treated. MAIN OUTCOME MEASURES--Culture of urine and determination of C reactive protein concentration every six weeks for the first six months after diagnosis, every three months from six months to two years, and every six months between two and three years; thereafter yearly urine culture. Evaluation of renal concentrating capacity with a desmopressin test; radiological examination, including first and follow up urography and micturition cystourethrography without antibiotic cover; and measurement of renal parenchymal thickness and renal surface area. RESULTS--Of the original 50 infants, 37 (12 girls, 25 boys) were followed up for at least six years. Two infants developed pyelonephritis within two weeks after bacteriuria was diagnosed; the others remained free of symptoms. 45 Infants were untreated; the bacteriuria cleared spontaneously in 36 and in response to antibiotics given for infections in the respiratory tract in eight. Recurrences of bacteriuria were observed in 10 of the 50 children, of whom one had pyelonephritis. No child had more than one recurrence. At follow up urography in 36 of the 50 children (9 girls, 27 boys) after a median of 32 months no child had developed renal damage. First samples tested for renal concentrating capacity showed significantly higher values than those from a reference population (mean SD score 0.50, 95% confidence interval 0.21 to 0.79; p less than 0.001), but the last samples showed no significant difference (mean SD score 0.08, -0.24 to 0.40; p greater than 0.05). CONCLUSIONS--Mass screening for bacteriuria in infancy results primarily in detection of innocent bacteriuric episodes and is not recommended.     

Linkage of the DNA-segment D7S13 (pB79a) with the cystic fibrosis locus

Summary A map distance of 2.9 cM between D7S13 (pB79a) and the cystic fibrosis (CF) locus was obtained from the analysis of 13 informative families with a history of CF. This result is based solely on the HindIII restriction fragment length polymorphism (HindIII-RFLP) at D7S13, since the interpretation of the Msp1-RFLP at this locus was found to be unreliable.     

In vivo formation of omega-oxidized metabolites of leukotriene C4 in the rat

[3H8]Leukotriene C4 was administered to germfree rats and to conventional rats having a bile duct cannula. Several radioactive metabolites were isolated. Two polar biliary metabolites from conventional rats were identified as N-acetyl-omega-carboxy-leukotriene E4 and N-acetyl-omega-hydroxy-leukotriene E4. A polar fecal metabolite from germfree rats was found to be N-acetyl-omega-carboxy-leukotriene E4. Chemical identities were established using UV spectroscopy and cochromatographies with authentic compounds in several HPLC systems. The fecal metabolite was further characterized by reductive desulfurization followed by gas-liquid-radiochromatography. The yield of the two biliary metabolites was 5% of the administered tritium after three hours and the yield of fecal N-acetyl-omega-carboxy-leukotriene E4 was 3.5% after three days.     

The nucleotide sequences of barley cytoplasmic glutamate transfer RNAs and structural features essential for formation of δ-aminolevulinic acid

In chloroplasts and a number of prokaryotes, -aminolevulinic acid (ALA), the universal precursor of porphyrins, is synthesized by a multistep enzymatic pathway with glutamyl-tRNA^Glu as an intermediate. The ALA synthesizing system from barley chloroplasts is highly specific in its tRNA requirement for chloroplast tRNA^Glu; a number of other Glu-tRNAs are inactive in ALA formation although they can be glutamylated by chloroplast aminoacyl-tRNA synthetases. In order to obtain more information about the structural features defining the ability of a tRNA to be recognized by the ALA synthesizing enzymes, we purified and sequenced two cytoplasmic tRNA^Glu species from barley embryos which are inactive in ALA synthesis. By using glutamylated tRNAs as a substrate for the overall reaction, we showed that Glu-tRNA reductase is the enzyme responsible for tRNA discrimination.