[Efficiency of the ALL-MB-2002 protocol in children with acute lymphoblastic leukemia].

AIM: To evaluate the efficiency of the original ALL-MB-2002 protocol within the multicenter study of treatment of acute lymphoblastic leukemia (ALL) in children. SUBJECTS AND METHODS: A total of 1873 primary patients with ALL aged 1 to 18 years, of whom 1544 patients were enrolled in this study, were notified at 36 clinics of Russia and Belarus from April 15, 2002, to January 1, 2008. RESULTS: With the median observation of 4.12 years, 7-year event-free survival (EFS) was 73 +/- 13%; overall survival (OS) 78 +/- 2%; relapse-free survival 82 +/- 1%. The rates of EFS and OS were equal and amounted to 76 +/- 2 and 80 +/- 2% in the standard-risk group (SRG) and intermediate-risk group (ImRG), respectively. In the high-risk group (HRG) patients, EFS and OS were as high as 30 +/- 6 and 37 +/- 6%, respectively. The frequency of relapses with central nervous system lesion was as much as 4.7% in all the patients, 6-year cumulative risk for isolated neurorecurrences being 2.5% in the SRG patients. Adolescents, patients with the baseline leukocytosis (more than 100 x 10(9)/l), and those with a splenic size of over 4 cm or more from the costal arch margin had substantially worse survival rates. A poor early response to therapy (on induction days 8 and 15) was also associated with its lower efficiency. CONCLUSION: Despite a considerable rise in the number of centers and a slight increase in the intensity of therapy, the results of the new ALL-MB-2002 protocol are as minimum equivalents obtained in the use of the previous ALL-MB-91 protocol. A significant improvement in the overall results of therapy and a reduction in the cumulative risk for isolated neurorecurrences were noted in the ImRG patients.

[Variation in mitochondrial DNA in Far Eastern mullet pilengas, Liza haematocheilus Temminck and Schlegel, acclimatized in the Azov-Black Sea basin].

This study continues the investigation of genetic variation in the populations of native and acclimatized in the Azov-Black Sea basin pilengas from the Sea of Japan. The previous comparison based on allozyme analysis was supplemented by analysis of restriction polymorphism of a mitochondrial DNA fragment containing the cytochrome b gene and the D-loop. Five out of fifteen endonucleases tested detected polymorphic sites. In the samples of native and acclimatized pilengas, five common haplotypes were found; ten and three "population-specific" haplotypes were detected in the Far Eastern and the Azov populations, respectively. The differences in haplotype distributions between these populations were highly significant (P < 0.001). The mtDNA variation was lower in the Azov than in the Far Eastern population (haplotype diversity mu respectively 6.35 +/- 0.27 and 9.14 +/- 0.55), which is in good agreement with the decrease in the number of polymorphic loci and the mean number of alleles per locus, found earlier for allozyme markers in this population. The reasons for these differences in the acclimatized population are discussed.

[The results of a multicenter trial of acute lymphoblastic leukemia treatment on ALL-MB 91/ALL-BFM 90m in children: analysis of efficacy and toxicity].

AIM: A comparative analysis of efficacy and toxicity of two chemotherapy regimens: standard German protocol ALL-BFM 90m and less intensive original test protocol ALL-MB 91 in a multicenter trial of acute lymphoblastic leukemia (ALL) in children. MATERIAL AND METHODS: In 1995-2002 a total of 834 patients with newly diagnosed ALL aged 0-18 years were admitted to 10 clinics of Russia. Of them, 713 were randomized in two groups: treatment program ALL-BFM 90m (n = 355) and ALL-MB 91 program (n = 358). RESULTS: In 7-year follow-up median, 10-year event-free survival (EFS) and overall survival (OS) did not differ significantly between the groups and was 67 +/- 3 and 68 +/- 3% (ALL-MB 91) and 74 +/- 2, 71 +/- 3% (ALL-BFM 90m), respectively. Though the rate of isolated recurrences in CNS in patients on the protocol ALL-MB 91 was 2.8%, they developed only in 0.8% patients of the standard risk group. Anemia, thrombocytopenia and agranulocytosis developed less frequently, hospital stay was significantly shorter on the test protocol vs the control one (p < 0.01). CONCLUSION: EFS and OS on the test (ALL-MB 91) and control (ALL-BFM 90m) protocols were equivalent in lower toxicity and cost of therapy.

Spectral properties of phosphopyridoxyl-Lys-7(-41)-ribonuclease A.

We have studied the spectral properties of RNAase A containing a phosphopyridoxyl residue at the epsilon-NH2 group of Lys-7 or Lys-14. The overall conformations of the native and modified enzymes were shown to be rather similar. All three proteins have similar circular dichroism spectra within the 220-300-nm region, and similar thermal transition temperatures. All the changes in the RNAase A molecule modified are located in close proximity to the alkylated lysine residue. The phosphopyridoxyl group of (P-Pxy)-epsilon-Lys-41-RNAase A is situated directly at the enzyme active site and is 25% butied in the protein globule. The P-pyridoxyl group of (P-Pxy)-epsilon-Lys-7-RNAase A was shown to be located in the vicinity of the active site and to be more exposed to the solvent. In the pyridoxyl phosphate absorption band, optical activity is induced in both proteins. Study of the pH dependence of the changes occurring in the circular dichroism and absorption spectra has shown that in the modified proteins, the pyridoxyl phosphate chromophore is rather sensitive to the ionic state of the surrounding medium and serves as a "reporter" group when the relationship between structure and function of the RNAase A active site is being investigated.

1,4-Dihydropyridine receptor associated with Ca2+ channels in human embryonic fibroblasts.

By using the radioactively labeled 1,4-dihydropyridine (DHP) probe, [3H]PMD, we have demonstrated that cultured human embryonic fibroblasts grown at a low density in Eagle's medium supplemented with serum contain a single class of non-interacting DHP binding sites (Bmax, 1.2 +/- 0.3 pmol/10(6) cells; Kd, 3.9 nM). After inhibition of the DHP receptor biosynthesis by cycloheximide, the number of [3H]PMD binding sites is reduced with a half-time of 12 h, which implies a turnover rate of 30,000 +/- 7500 receptors/h per cell. With progression to confluency, the Bmax value decreased up to 0.28 +/- 0.08 pmol/10(6) cells without significant change in Kd value. When cells were grown at a low density in serum-free conditions, the number of [3H]PMD binding sites gradually increased 1.9-fold within 3 days. Addition of serum reversed this effect with the same time course. These results imply that the DHP-sensitive Ca2+ channels are involved in the control of the proliferation of human embryonic fibroblasts.

Calcium entry blockers and oxiracetam have opposite effects on the density of dihydropyridine receptors in rat cerebral cortex.

Ca2+ entry blockers riodipine, D-cis-diltiazem and verapamil, when administered i.p. to rats at a dose of 10 mg/kg, produced two-fold decreases in the density of 1,4-dihydropyridine (DHP) receptors in rat cerebral cortex, as revealed by Scatchard plot analysis of radioligand binding made 24 h after the first injection. Thereafter, the number of DHP binding sites increased up to the initial level on day 4 of the treatment. The nootropic drug oxiracetam, when injected simultaneously with Ca2+ channel blockers at a dose of 10 mg/kg, prevented this transient decrease in DHP receptor density in brain. These results can explain the opposite modulation of memory retention by calcium antagonists and nootropic drugs that has been observed previously.

Mechanisms of cyclic AMP phosphodiesterase regulation.

The mechanisms of regulation of cyclic AMP phosphodiesterases were studied using the cytoplasmic fraction of PC-12 cells sensitive to the action of nerve growth factor. The cells contain phosphodiesterases of two types. One of them possesses a high affinity for cyclic AMP (Km = 2.46 mM), whereas the other has the affinity by an order worse (Km = 37.1 mM). PC-12 cell differentiation under the action of nerve growth factor is connected with the cyclic nucleotide elevation; however, activities of both phosphodiesterases remain unchanged. This indicates that the regulation of activity of these enzymes in PC-12 cells is mediated by second messenger effects. The main object of cell regulation is phosphodiesterase with low affinity for the substrate. Its activity is modulated by the calmodulin-Ca2+ complex, cyclic GMP and NAD+ at micromolar concentrations. The effect on the phosphodiesterase system of both a "quick" messenger, Ca2+ and "slow" messengers, cyclic GMP and NAD+, has the same consequences: the turnover number of the enzymic reaction increases that is accompanied by a proportional decrease in the enzyme affinity for cyclic AMP so that the ratio Vmax/Km remains constant. A possible explanation of functional significance of such an activity modulation may be the necessity to maintain the conditions for phosphodiesterase functioning when Km much greater than [cyclic AMP] and the reaction rate are directly proportional to the substrate concentration: v = Vmax/Km [cyclic AMP]. Then the cells are transferred into such a mode when autoregulation of the cyclic nucleotide level takes place. Besides the transient effects causing changes in phosphodiesterase activity, studies of PC-12 cells revealed a chronic effect of phosphodiesterase activity change under the action of staphylococcal enterotoxin A. This protein which induces differentiation of PC-12 cells and possesses a NAD+-glycohydrolase activity is translocated into cytoplasm of cells in the presence of NAD+ and accomplishes ADP-ribosylation of phosphodiesterase. As a result, the enzyme activity falls, cyclic AMP level increases and cell differentiation starts. The activity of soluble phosphodiesterase of PC-12 cells also decreases under the effect of two neurotoxins from bee venom, melittin and tertiapin. Both the toxins at concentration of 10 microM completely block calcium regulation of the enzyme. The mechanism of tertiapin action was investigated on a model system of calmodulin-bovine brain phosphodiesterase. It appeared that inhibition of Ca2+ action is achieved as the result of binding of two toxin molecules with Kd = 2 mM to the activated calmodulin molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

[Role of the ribose residue of substrates in reactions catalyzed by ribonuclease]. / Rol' riboznogo ostatka substratov v reaktsiiakh, kataliziruemykh ribonukleazami.

The rate constants and Km for the hydrolysis of the optically active nonglycosidic analogues of the CpA and C greater than p catalysed by RNase A and RNase BS-I were measured. The rate of hydrolysis of the model substrates in 10(5) and 10(3) slower that for the appropriate dinucleoside phosphate and nucleoside cyclophosphate. However, substitution of the relatively rigid ribofuranose ring with flexible alifatic chains is accompanied by little variation in binding constants. The analyses based on the single substrate system indicate that the observed difference in rate constants must be accounted for by a difference between the binding of the substrates in the transition state to the RNase active site. Consequently, the "rigidity" of the ribose rings in RNA leads to large decreases in the free energy of activation for the reactions catalysed by RNases.

[Steady state kinetics of DNA degradation with pancreatic deoxyribonuclease A in the presence of Mg2+]. / Statsionarnaia kinetika passhchepleniia DNK pankreaticheskoi dezoksiribonukleazoi A v prisutstvii Mg2+.

Steady state kinetics of DNA depolymerisation in the presence of the DNAase A and Mg2+ ions were investigated at pH 5.5 and wide region of the enzyme, substrate and metal ion concentrations. A model, which is consistent with experimental results obtained is suggested. According to the model catalytically active form of the DNAase A should be a metal-bound enzyme. That species reacts with the metal-free DNA to form the Michaelis complex. The kinetics observed can be described in terms of mechanism which involves covalent enzyme-substrate intermediate formation. It was shown that the second Mg2+ ion binding to the complex Mg2+ DNAase -- DNA (KD - 2.2 . 10(-3) M) enhances the kinetic parameters of the reaction. To rationalise the effect one has to assume that the rate of the intermediate formation was accelerated as a result of the second Mg2+ binding.

[Problems of cellular activity regulation by secondary messengers]. / Problemy reguliatsii kletochnoi aktivnosti posredstvom vtorichnykh messendzherov.

The paper surveys the data obtained during the last years in the Laboratory of Regulation of Cellular Activity of the Institute of Molecular Biology. The data mostly concern the enzymes taking part in metabolism and in biological action of the secondary messengers, as well as the mechanisms of regulation of some metabolic pathways in the cultured cells. The results pertinent to the mechanisms of chromatin condensation and based on this genetic control processes are also discussed.

[Conformational stability of ribonuclease A complexes with specific inhibitors]. / Konformatsionnaia stabil'nost' kompleksov ribonukleazy A so spetsificheskimi ingibitorami.

Isotermic unfolding of ribonuclease A, phosphopyridoxyl-8Lys41-RNAase A and complexes of the enzyme with cytidine, 2'-CMP, 3'-CMP, 3'-AMP and with the phosphoric ester of 1-(omega-oxypropyl)-cytosine in presence of urea has been studied. The stabilization of the protein structure resulting from the complex formation was shown to be determined by the ligand nucleobase binding. The comparison of the results obtained with those known from the literature suggests, that binding and catalytic zones of the enzyme active site form an integrated network system which is substained by multipoint contacts between the constituents. The change in the state of any part within the enzyme active state affects the energetics of the whole protein globule.

[Physico-chemical properties of ribonuclease A modified with pyridoxal-5'-phosphate]. / Fiziko-khimicheskie svoistva ribonukleazy A, modifitsirovannoi piridoksal'-5'-fosfatom

The physico-chemical properties have been studied of RNase A selectively modified at the E-NH2-group of Lys-7 and Lys-41 with pyridoxal-P. Modification did not affect conformational stability of the protein globule, thus all changes in the molecule of the modified RNase A were localised around the alkylated Lys residue. In the both cases pyridoxyl-P. The residue was shown to be localized in the active site region of the (P-Pxy)-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. (P-Pxy) E-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. In the Lys-41 derivative, pyridoxamine-P was situated exactly in the active site and is partially hidden in the protein grobule. The pH-dependence of absorption spectra indicates that the chromophore of pyridoxyl-P in modified proteins is quite sensible to the ionic state of its surrounding. The usefulness of pyridoxyl-P as a reporter group was proved in the study with (P-Pxy)-Lys-7-RNase A. Some conformational changes involving His-119 were shown to take place in the course of the enzyme-nucleotide complex formation.

[Primary structure of calmodulin from the human brain]. / Pervichnaia struktura kal'modulina iz mozga cheloveka.

The primary structure of calmodulin from human brain has been determined. As compared to calmodulin from bovine brain, it shows three substitutions of the amide----acid type at positions 24, 60, 129, and two acid----amide changes at positions 104 and 135.

[A receptor of dihydropyridine blockaders of Ca2+ influx in human embryonic fibroblasts]. / Retseptor digidropiridinovykh blokatorov vkhoda Ca2+ v émbrional'nykh fibroblastakh cheloveka.

The plasma membrane of human embryonic fibroblasts was shown to contain receptor binding sites for 1,4-dihydropyridine (DHP) Ca2+ entry blockers. In a subconfluent culture grown in serum medium the content of the DHP receptor amounted to 1.2 +/- 0.3 pmol per 10(6) cells. With progression to confluency this value decreased up to 0.28 +/- 0.08 pmol per 10(6) cells. The DHP binding capacity was shown to be affected by the presence of growth factors in culture medium. In a subconfluent culture of serum-deprived cells the content of DHP binding sites increased 1.9 fold, the steady-state level being achieved within 3 days in culture. The serum gradually reversed this process, and the DHP receptor density approached the initial level within 3 days.

[Far Eastern mullet Mugil soiuy Basilewsky (Mugilidae, Mugiliformes): the genetic structure of populations and its change under acclimatization]. / Dal'nevostochnyi pilengas Mugil soiuy Basilewsky (Mugilidae, Mugiliformes): geneticheskaia struktura populiatsii i ee izmeneniia pri akklimatizatsii.

The introduction of Far Eastern mullet (pilengas) in the Azov Sea in the 1970s-1980s has resulted in the formation of a self-reproducing commercial population. We have carried out a comparative population-genetic analysis of the mullet from the native (Primorye, the Sea of Japan basin) and the new (The Azov Sea basin) ranges. Genetic characteristics of three Primorye and three Azov local samples were studied using electrophoretic analysis of 15 enzymes encoded by 21 gene loci. In the Azov mullet, the initial heterozygosity characteristic of the donor population was preserved while the genotype and the allele compositions changed; the changes included a 1.9-fold reduction in the percentage of polymorphic loci and 1.5-fold reduction in the mean number of alleles per locus. The genetic differences between the Azov and the Primorye sample groups were highly significant. In the native range, no genetic differentiation among the mullet samples from different areas was found (Gst = 0.42%), whereas in the Azov Sea basin, the samples from spatially isolated populations (ecological groups) exhibited genetic differences (Gst = 1.38). The genetic divergence of the subpopulations and the excess of heterozygotes at some loci in the Azov mullet suggest selection processes that formed genetically divergent groups associated with the areas of different salinity in the new range. The salinity level is assumed to be the most probable factor of local differentiating selection during fast adaptation and naturalization of the introduced mullet.

[Biochemical estimation of individual sensitivity to oxygen intoxication in rabbits]. / Biokhimicheskaia otsenka individual'noi chuvstvitel'nosti k kislrodnoi intoksikatsii u krolikov.

Correlation between a pro-oxidant activity of blood, estimated by means of chemoluminescence in the system H2O2-luminol-blood plasma, and individual sensitivity of rabbits to the effect of oxygen was studied. Alterations in the blood pro-oxidant activity, as shown by treatment of the blood sample with 0.7 MPa of oxygen in vitro, correlated distinctly with the period of convulsions as well as with viability of animals during acute hyperoxia.

[Results of the treatment of childhood acute lymphoblastic leukemia according to the Berlin-Moscow-91 protocol in 1991-2000]. / Rezul'taty lecheniia ostrogo limfoblastnogo leikoza u detei po protokolu Moskva-Berlin-91 v 1991-2000 gg.

The report deals with the results of application of an original protocol--the Berlin-Moscow-91 (BM-91)--for the treatment of acute lymphoblastic leukemia (ALL) in children. The researchers' major concern was to improve survival and cut down side-effects incidence as well as to prevent and successfully manage occult neuroleukemia as a potential source of relapse. Patients aged 5 months-15 years received the BM-91 and ALL BFM-90m treatment first at one clinic and later at several centers. Out of 852 children with primary diagnosis of ALL admitted to Russian hematological hospitals (March 2, 1991-November 3, 2000), 687 were included into the study; 329 received the MB-91 protocol. Nine-year recurrence-free survival was 73% while overall survival--80%. Toxic side-effects after L-asparaginase were reported in 27 (7.9%). It is concluded that good results in childhood ALL treatment can be achieved without resorting to high-dosage chemotherapy and radiation in most cases.

[Treatment of acute lymphoblastic leukemia in children according to the ALL-BFM-90m protocol in the Russian Federation and the Republic of Belarus]. / Lechenie ostrogo limfoblastnogo leikoza u detei po protokolu OLL-BFM-90m v Rossiiskoi Federatsii i Respublike Belarus'.

Prognosis for children treated according to the BFM-90m protocol (Berlin-Frankfurt-Munster Group) for acute lymphoblastic leukemia (ALL) improved significantly as compared with previous modalities. Methotrexate was used in the dose of 1,000 mg/m2, 36 h. The paper presents the 10-year results for this modification. Patients aged 0-15 years were treated at hematological hospitals of Moscow, other Russian towns and in Minsk, Belarus, (July 5, 1990-November 11, 2000). BFM-90m treatment was given to 682 children out of 1,326 with primary diagnosis of ALL; a comparative trial of the MB-91 protocol hed been carried out at the same clinics since 1991. During 10 years, recurrence-free survival was 72% while overall survival--77%. Toxicity of side-effects was tolerable. The BFM-90m treatment showed significantly better results in both countries.