High frequency of RAS oncogene mutation in chronic myeloid leukemia patients with myeloblastoma.

To determine the role of the mutated RAS oncogene during development into the blast phase, we sequentially analysed RAS oncogene mutations in the bone marrow of 27 patients with chronic myeloid leukemia (CML). DNA from CML patients in chronic and blast phases and nude mouse tumor DNA formed by a tumorigenicity assay (in vivo selection assay) were subjected to the polymerase chain reaction (PCR) and oligonucleotide hybridization. In addition, one patient in the chronic phase and five in the blast phase were also analysed. PCR analysis of DNA from the leukemic patients revealed that 3.6% (1 of 28) and 15.6% (5 of 32) of the patients in the chronic and blast phases, respectively, had RAS mutations. N- or K-RAS oncogene mutations were found mostly in the blast phase (4 of the 5 patients with the RAS oncogene mutation). Of the 5 patients with the RAS oncogene mutation, three developed myeloblastoma, a myeloblast cell tumor, in the blast phase. None of the 28 patients without the RAS mutation developed myeloblastoma. These results suggest that the RAS oncogene mutation occurred in the late stage of the disease and contributed to transformation to the blast phase in some CML patients. The findings also indicate an association between the presence of the RAS mutation and the formation of myeloblastoma.

Telomere reduction of specific chromosome translocation in acute myelocytic leukemia.

The length of telomere restriction fragments (TRF) was studied by Southern blotting in 42 patients with acute myelocytic leukemia (AML) including 15 patients with 8;21 translocation, 8 with 15;17 translocation and 19 with a normal karyotype. The TRF length of leukemic cells with a normal karyotype and with 8;21 or 15;17 translocation showed significant reduction compared to that of lymphocytes from normal individuals (P < 0.05). In addition, there was a statistically significant difference in TRF length between leukemic cells with a normal karyotype and 8;21 translocation (P < 0.05). Therefore, the significant difference of telomere reduction in 8;21 translocation may be an important factor in the leukemogenic process.

Cytogenetic 2; 18 and 18; 22 translocation in chronic lymphocytic leukemia with juxtaposition of bcl-2 and immunoglobulin light chain genes.

The majority of follicular lymphoma cells carry the typical chromosome translocation 14;18, which juxtaposes the bcl-2 gene to the immunoglobulin heavy-chain (IgH) gene. Variant translocations of the bcl-2 gene to the Ig lambda or Ig kappa gene have been found by molecular biological techniques in a significant fraction (approximately 10%) of chronic lymphocytic leukemia (CLL). However, there have been no reports describing the presence of cytogenetic 18;22 and 2;18 translocations in CLL, in spite of extensive karyotypic studies. We present here two cases of CLL, one with cytogenetically detected t(2;18)(p11;q21) and the other with the t(18;22)(q21;q11). The molecular analysis revealed that these translocations juxtaposed the bcl-2 and immunoglobulin light-chain (IgL) genes. The t(18;22) broke the 5' flanking region of the bcl-2 gene and juxtaposed to the immunoglobulin lambda light-chain (Ig lambda) gene in a head-to-head configuration, as in the cases previously described. In the case of the t(2;18), the bcl-2 gene and immunoglobulin kappa light-chain (Ig kappa) gene were juxtaposed in a head-to-tail configuration, which is opposite to that expected from the orientation of the genes on chromosomes. The breakpoint was located within the 5' untranslated region of the bcl-2 gene. The results presented here indicate that the bcl-2/immunoglobulin light-chain (IgL) gene juxtaposition seen in a fraction of CLL is the result of cytogenetically detectable reciprocal chromosome translocations 2;18 and 18;22.

[The significance of cytogenetic analysis in the diagnosis of leukemia].

The results of a comprehensive study on 1,000 patients who had been cytogenetically diagnosed to have leukemia in our department since 1962 are reported, and the value of cytogenetic diagnosis for leukemia emphasized. In our series, we detected patients with FAB L1 and L2 showing an abnormality rate of 60 and 66%, respectively. They included 20% with Ph1 positive ALL. In FAB L3, we found t(8;14) in 5 of the 6 patients. The FAB M1 group showed the lowest abnormality rate (50%). Forty percent of the M2 patients exhibited t(8;21), 60% of which also showed loss of sex chromosome of either X or Y. Seventy-eight percent of the M3 patients presented t(15;17) (two patients with no detectable t(15;17) showed rearrangements of retinoic acid receptor alpha gene). Inversion of chromosome 16 was found in 10% of the patients with FAB M4. Patients with M6 exhibited relatively complex chromosomes aberrations. RAEB patients showed more frequent and complex type of chromosome aberrations than PARA patients. Cytogenetic and molecular-biological analyses provided valuable information on the pathophysiology of leukemia and suggested the possible localization of novel oncogene(s).

Novel molecular analogues of phosphatidylcholines in a lipid extract from bovine brain: 1-long-chain acyl-2-short-chain acyl-sn-glycero-3-phosphocholines.

A vasodepressor phospholipid fraction named Depressor-IA from a bovine brain lipid extract was analyzed by capillary gas-liquid chromatography-mass spectrometry as tert-butyldimethylsilyl derivatives after hydrolysis with phospholipase C. Results show that Depressor-IA is a mixture of 3 platelet-activating factors and 17 1-acyl analogues. Three platelet-activating factors having sn-1-O-hexadecyl, octadecyl, and octadecenyl groups were suggested to account for the hypotensive activity of Depressor-IA, although the total amount of 1-acyl analogues of platelet-activating factor was much more than that of platelet-activating factor in the purified Depressor-IA. 1-Long-chain acyl-2-short-chain acyl-glycero-3-phosphocholines identified in Depressor-IA included novel molecular analogues having sn-2-propionyl, acryloyl, butyryl, valeryl, caproyl, and hepatonoyl groups.

Theoretical analysis of a site-specific chemiluminescence reaction and its application to quantitation of lipid hydroperoxides.

A system was designed for chemiluminescent measurement of lipid hydroperoxides by their site-specific reaction in sodium dodecylsulfate micelles. Ferrous ion-induced decomposition of lipid hydroperoxides in the sodium dodecylsulfate micelles resulted in strong chemiluminescence of the Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (CLA). After addition of ferrous sulfate to the micelles containing lipid hydroperoxide and luciferin, the chemiluminescence intensity reached a maximum rapidly and then decreased. The sequence of this reaction was elucidated by theoretical analysis, which demonstrated that the maximum chemiluminescence intensity is proportional to the initial concentration of hydroperoxide. Good linear relationships were observed between the maximum counts of chemiluminescence and the amounts of hydroperoxides of linoleic acid, phosphatidylcholine, choresterol (5 alpha), cumene and tert-butyl and hydrogen peroxide. This chemiluminescence method was simple and sensitive enough to detect picomole levels of linoleic acid and phosphatidylcholine hydroperoxides.

Novel phospholipids with aliphatic dicarboxylic acid residues in a lipid extract from bovine brain.

A vasodepressor phospholipid fraction purified from a lipid extract of bovine brain was found to contain novel phospholipids with both a long-chain acyl group and an aliphatic dicarboxylic acid residue. This was shown by analyzing the fraction as tert-butyldimethylsilyl derivatives of glyceride by capillary GC-MS after hydrolysis with phospholipase C. Six molecular species with a palmitoyl group and an aliphatic dicarboxylate (chain length C4-C9), and two species with both a stearoyl group and a succinate or glutarate residue were detected.

Evidence for existence of various homologues and analogues of platelet activating factor in a lipid extract of bovine brain.

Vasodepressor phospholipid with platelet-aggregating activity was highly purified from a lipid extract of bovine brain and subjected to field desorption-mass spectrometry. It was further analyzed by gas-liquid chromatography-mass spectrometry after hydrolysis with phospholipase C and conversion to tert-butyldimethylsilyl derivatives. Results indicated the presence of four species of platelet activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) and ten acyl analogues of PAF. The acyl analogues of PAF included species having an sn-2-propionyl or sn-2-butyryl group, which have not been previously detected in natural sources. The total amount of acyl analogues of PAF was much higher than that of PAF.

A platelet-aggregating and hypotensive phospholipid isolated from bovine brain.

A phospholipid that differs from known active lipids and causes potent platelet aggregation and weak hypotension has been isolated from bovine brain. Its platelet aggregating effect on heparinized platelet-rich plasma from rabbits, was at a threshold concentration of about 0.2 nmol ml-1 as phosphorus. The effect was inhibited by CV-3988. The phospholipid was converted by diazomethane treatment to another active lipid that caused short-term hypotension, but not platelet aggregation, rather it inhibited the aggregation of rabbit heparinized platelets induced by platelet-activating factor.

Isolation of diosgenin[(25R)-spirost-5-en-3 beta-ol] from a lysate of a new hypotensive phospholipid occurring in bovine brain.

Diosgenin[(25R)-spirost-5-en-3 beta-ol] and cholesterol were detected as trimethylsilyl derivatives by gas chromatography-mass spectrometry in an alkaline lysate of a new hypotensive phospholipid from the lipid fraction of bovine brain. These steroids seemed to be present in the hypotensive phospholipid fraction in some bound forms.

Phosphorus-free antihypertensive lipid from dog peritoneal dialysate, Peritoneal Dialysate Depressor-II.

A phosphorus-free lipid with a transient antihypertensive effect was found in a dog peritoneal dialysate by a modified procedure. On silicic acid chromatography of the crude extract, a new hypotensive factor and a new hypertensive factor were separated besides the known antihypertensive phospholipid resembling lysolecithin (Peritoneal dialysate depressor-I). The new antihypertensive factor was purified almost completely by gel filtration on a Sephadex LH-20 column, and found to contain no phosphorus. It was tentatively named Peritoneal Dialysate Depressor-II. The main spot detected on preparative thin-layer chromatography by charring with ethanolic sulfuric acid reagent was found to be responsible for the antihypertensive action. This factor caused a sharp decrease in systemic arterial blood pressure of rats, cats and guinea pigs when injected into the femoral vein. There was no significant change in the heart rate during this hypotension. The hypotensive activity was not affected by previous treatment of the rats or guinea pigs with antimuscarinic, antihistaminic, beta-adrenergic-blocking or ganglionic-blocking agents. The antihypertensive effect of the factor was unaffected on rats pretreated with yohimbine, indomethacin or theophylline. In spinal or reserpinized rats, no detectable reduction of the depressor activity was observed. The factor did not affect isolated guinea pig ileum. Its depressor activity was not changed significantly by its treatment with proteases. No prostaglandin E or F series compounds were detected in the preparation by fluorometric assay with 15-hydroxyprostanoate oxido-reductase and resazurin. The hypotensive factor caused no aggregation of cat or rabbit platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of antispasmodic effect of synthetic lysolecithins with various fatty acid moieties on guinea pig ileum.

Non-competitive antispasmodic effects of 8 kinds of synthetic L-alpha-lysolecithins (1-O-acyl-2-lyso-sn-glycero-3-phosphocholine, LPC) with various fatty acid moieties were examined upon the spasmodic actions of histamine and acetylcholine on guinea pig ileum. Five-min pretreatment of the gut with the synthetic LPC was required to be effective. With increase of the concentration of a synthetic LPC for pretreatment of the ileum, the slope of the dose-response curve of histamine or acetylcholine added cumulatively became more gentle, the maximal contraction was suppressed apparently, and which were associated with the shift of the curve to the higher concentration of the stimulants. Of LPCs with saturated fatty acid palmitoyl-LPC showed the strongest effect, followed by myristoyl-, stearoyl-, lauroyl- and decanoyl-LPCs in order. Incorporation of cis-double bond into the C18 fatty acid chain of LPC resulted in slight decrease of the antispasmodic effect. The relaxating effect of LPCs on perfused rabbit ear vessel preparations was similar in order.

Cardiovascular effect of "peritoneal dialysate-depressor-I", a short-active hypotensive phospholipid in dog peritoneal dialysate.

Cardiovascular action of "peritoneal dialysate-depressor-I" (PD.D-I), a short-acting hypotensive phospholipid occurring in dog peritoneal dialysate, was investigated. With an intravenous injection of PD.D-I into an anaesthetized rat, a sharp fall of arterial blood pressure was observed and the effects were dose dependent. The maximum hypotensive effect was about 60 mmHg and the minimum effective dose was approximately 35 micrograms/kg. Neither tachyphylaxis nor sensitization was observed. Even in conscious rats PD.D-I elicited hypotensive responses, though the effect was much weaker than that produced in anaesthetized rats. In spinal rats the hypotensive effects were also observed. In the tests on rats reserpinized or pretreated with antimuscarinic, antihistaminic, beta-adrenergic-blocking and ganglionic-blocking agents, the depressor effect of PD.D-I was not affected. PD.D-I elicited also hypotensive responses in anaesthetized cats, rabbits and guinea pigs in the same degree as those in anaesthetized rats. The relaxation of the peripheral blood vessels was observed in the test on perfused rabbit ear. The depressor factor showed no smooth muscle stimulating activity in isolated guinea pig ileum preparations. Judging from these findings, the hypotensive effect of PD.D-I is not ascribable to the central, sympathetic or parasympathetic nervous system but possibly to direct action on the peripheral vascular system.

Occurrence of short-duration hypotensive phospholipid from dog peritoneal dialysate.

From the total lipid fraction of dog peritoneal dialysate after freeze drying and extraction, a hypotensive phospholipid was isolated through silicic acid, cellulose, and Sephadex LH-20 column chromatography in a pure grade; it showed a single spot on TLC. The purified hypotensive factor, designated as Peritoneal Dialysate Depressor-I, elicited potent depressor responses in anesthetized rats, and its threshold dose was approximately 35 microgram/kg. The material resisted proteases and 15-hydroxyprostanoate oxidoreductase. In mobility on TLC, the hypotensive factor was distinuishable from water-soluble hypotensive substances and also from depressor lipids. Judging from its behavior on TLC and column chromatography during the purification procedure, the hypotensive factor seems to be a choline-containing phospholipid and shows the general characteristics of hysolecithin, except for its potent hypotensive activity, and 2',7'-dichlorofluorescein on TLC. The molar ratio of phosphorusurated ones such as stearic and palmitic acids.