The use of autologous peripheral blood stem cells as a hemopoietic support during polychemotherapy of children with soft tissue sarcomas.

Intensification of cycle polychemotherapy in disseminated tumors considerably improves the efficiency of complex treatment. Reinfusion of peripheral blood stem cells as a factor of replacement treatment during hemopoietic suppression or disorders is now becoming more and more promising.

[Innovation approaches to treatment of children with malignant tumors of the musculoskeletal system].

The Department of Musculoskeletal System Tumors was formed in November 1989 within Research Institute of Pediatric Oncology and Hematology of N. N. Blokhin Russian Oncological Scientific Center of Russian Academy of Medical Sciences. Treatment is carried out with the newest achievements in oncopediatrics applied; priority is given to limb-sparing techniques. The total 5-year survival rate of patients with Ewing's sarcoma is 65.6%, the total 2-year survival rate of patients with soft tissue sarcomas is 62.2%, and the total 2-year survival rate of children with osteosarcoma is 76%. Study of complications and side-effects of chemotherapy, as well as the development of methods of their prevention and correction, present a separate direction in the department's activity. In 1999, the team of the institution's researchers was awarded Russian Federation State Prize for outstanding scientific achievements in the work Development and Clinical Application of Combined Methods of Treatment of Osteogenic Sarcoma. In 2003 and 2004, leading researcher N. M. Ivanova was awarded the institution's diplomas as a part of Biotech prize for the development of new methods of treatment of malignant tumors in children.

Structural principles of the wide substrate specificity of Thermoactinomyces vulgaris carboxypeptidase T. Reconstruction of the carboxypeptidase B primary specificity pocket.

Site-directed mutagenesis in the active site of Thermoactinomyces vulgaris carboxypeptidase T (CpT), which is capable of hydrolyzing both hydrophobic and positively charged substrates, resulted in five mutants: CpT1 (A243G), CpT2 (D253G/T255D), CpT3 (A243G/D253G/T255D), CpT4 (G207S/A243G/D253G/T255D), and CpT5 (G207S/A243G/T250A/D253G/T255D). These mutants step-by-step reconstruct the primary specificity pocket of carboxypeptidase B (CpB), which is capable of cleaving only positively charged C-terminal residues. All of the mutants retained the substrate specificity of the wild-type CpT. Based on comparison of three-dimensional structures of CpB and the CpT5 model, it was suggested that the lower affinity of CpT5 for positively charged substrates than the affinity of CpB could be caused by differences in nature and spatial location of Leu247 and Ile247 and of His68 and Asp65 residues in CpT and CpB, respectively, and also in location of the water molecule bound with Ala250. An additional hydrophobic region was detected in the CpT active site formed by Tyr248, Leu247, Leu203, Ala243, CH3-group of Thr250, and CO-groups of Tyr248 and Ala243, which could be responsible for binding hydrophobic substrates. Thus, notwithstanding the considerable structural similarity of CpT and pancreatic carboxypeptidases, the mechanisms underlying their substrate specificities are different.

Thiol-dependent serine proteinase from Paecilomyces lilacinus: purification and catalytic properties.

An extracellular thiol-dependent serine proteinase was isolated from culture medium filtrate of the microscopic fungus Paecilomyces lilacinus with a yield of 33%. The enzyme is inactivated by specific inhibitors of serine proteinases, phenylmethylsulfonyl fluoride, as well as by chloromercuribenzoate and mercury acetate, but is resistant to chelating agents. The proteinase has broad specificity, hydrolyzes proteins and p-nitroanilides of N-acylated tripeptides, exhibiting maximal activity in hydrolysis of substrates containing long hydrophobic and aromatic residues (norleucine, leucine, phenylalanine) as well as arginine at the P1 position. The enzyme has a molecular weight of 33 kD. The enzyme is most active at pH 10.0-11.5; it is thermostable and is characterized by broad optimum temperature range (30-60 degrees C), displaying about 25% of maximal activity at 0 degrees C. The N-terminal sequence of the enzyme (Gly-Ala-Thr-Thr-Gln-Gly-Ala-Thr-Gly/Ile-Xxx-Gly) has no distinct homology with known primary structures of serine proteinases from fungi and bacilli. Based on its physicochemical and enzymatic properties, the serine proteinase from P. lilacinus can be classified as a thiol-dependent subtilisin-like enzyme.

[Modern prospects for polychemotherapy of solid neoplasms in children]. / Sovremennye vozmozhnosti polikhimioterapii solidnykh zlokachestvennykh opukholei u detei

To improve life quality and to increase survival in children with solid tumors, emphasis is placed on chemotherapeutical treatments along with other existing ones (surgery, radiation). By taking into account the fact that most malignant tumors in children are highly responsive to chemotherapy, programme treatment in children with neoplasms included the new drugs iphosphamide, vepeside, cisplatin, carboplatin, melfalan, bleomycin. The results of therapy with these drugs alone or in combination with well-known effective anthracycline antibiotics and plant preparations are presented. Whether more rigid modified schemes for administration and increment of dosage can be used is shown. This investigation opens new vistas for treating patients with not only localized, but disseminated forms of tumors, such as nephroblastomas, germinogenic tumors, soft tissue tumors, tumors of the bone, head, and neck. Regular assessment of the existing programmes in order to amend them, the use of new drugs, the increase of dosage and administration regimens enhance promises in treating malignant tumors in children.

[ES-IPO-97 treatment protocol for prognostically poor Ewing's sarcoma forms in children: results of implementation]. / Rezyl'taty provedeniia protokola ES-IPO-97 lecheniia detei s prognosticheski neblagopriiatnymi formami sarkomy Iuinga.

The paper shows the high efficiency and moderate toxicity of inductive treatment in children with Young sarcoma and primitive neuroectodermal tumors by ES-Ipo-97 protocol that includes alternate chemotherapy by the scheme: vincristine, 1.5 mg/m2/day, on days 1, 8, 15; adriamycin, 37.5 mg/m2/day, on days 1 and 2 as 24-hour infusion; cyclophosphanum, 2.1 g/m2/day, on days 1 and 2 (Block A); iphosphamide, 2.4 g/m2/day on days 1 to 5, etoposide, 100 mg/m2/day, on days 1-5 (Block B). It provides evidence for that this therapy is promising and awaits further developments.

[Ultrasound tomography in diagnosis of malignant soft tissue tumors in children]. / Ul'trazvukovaia tomografiia v diagnostike zlokachestvennykh opukholei miagkikh tkanei u detei.

Ultrasound findings of 50 children with soft tissue sarcomas of the trunk and limbs were analyzed. There was a high proportion of diagnostic errors (62%), which resulted in the choice of adequate therapeutical measures at early stages of therapy and in advanced disease. The reasons of the errors were as follows: nonspecificity of the first clinical manifestations of soft tissue sarcomas; ignorance of instrumental studies that establishes a diagnosis in time; outpatient physicians' unconcern of cancer, misunderstanding of the nontumor nature of disease; no scientifically grounded algorithm of studies. Echographic signs that characterize malignancy, their changes over a therapy, ultrasound pattern due to posttherapeutical changes are considered.

[Some indices of gastrointestinal tract status in children cured fro malignant neoplasms]. / Nekotorye pokazateli sostoianiia zheludochno-kishechnogo trakta u detei, izlechennykh ot zlokachestvennykh novoobrazovanii.

A total of 2255 individuals who cured from oncological diseases in childhood in Moscow in 1978 to 1997 have been studied. Most (60%) children undergone special treatment have serious visceral abnormalities. The authors examined the negative impact of a special treatment on the gastrointestinal mucosa and changes in its function after termination of special therapy in 79 cured patients. The most common diagnosed abnormalities included biliary dyskinesis (17-20%), dysbacterioris (16%), gastritis (7%). No abnormal gastrointestinal changes were detected in 37%.

[Radiologic diagnosis of malignant tumors of the pelvic bones in children]. / Rentgenologicheskaia diagnostika zlokachestvennykh opukholei kostei taza u detei.

An analysis of the data on radiodiagnosis of malignancies of pelvic bones in 50 patients, aged 10-15, is presented. Diagnosis was morphologically confirmed in all the cases: Ewing's sarcoma (41)--82%, primitive neuroectodermal tumor (6)--12% and chondrosarcoma (3)--6%. The leading clinical syndrome was pain in the loin. The significantly most frequent site was the iliac bone. X-ray imaging of tumor site and adjacent areas using standard direct, lateral and oblique projections, ultrasonography, X-ray contrast tomography, radioisotope examination, puncture and open biopsy (when indicated) proved useful.

[Diagnosis and therapy of rhabdomyosarcoma in children]. / Diagnostika i lechenie rabdomiosarkomy u detei.

An analysis of 24-month relapse-free survival in 62 patients with rhabdomyosarcoma (S.I.O.P.-89) is presented. All children with rhabdomyosarcoma (RMS) stage I have survived; stage II--90, and stage III--62%, i.e. twice as many as compared with those treated before 1993. RMS sites included retroperitoneal space, spatium perinei, urinary bladder, testicle, abdominal and thoracic cavity. Diagnosis was based on clinical, instrumental, laboratory, X-ray, radionuclide, ultrasound and morphological data. Recommendations for examination of RMS suspects were worked out for different levels of expertise.

[Therapeutic pathomorphism of Ewing's tumor]. / Terapevticheskii patomorfoz opukholi Iunga.

Pathomorphosis of Ewing's tumors after the combined treatment is studied with the use of light and electron microscopy, DNA-flow cytometry. Necrosis and apoptosis of tumor cells produced by the therapy are characterized. Therapeutic forms of tumor cells representing hyperaneuploid population delayed at the G2-level of the cell cycle are described.

[Clinical course, diagnosis and therapy of Ewing's sarcoma of the ribs in children]. / Klinika, diagnostika i lechenie sarkomy Iuinga reber u detei.

X-ray examination is considered to be the most effective procedure for diagnosis of Ewing's sarcoma of the rib. The extent of surgery and sequence of procedures are determined on the basis of its evidence. Treatment of infantile Ewing's sarcoma of the rib should include chemoradiation therapy and compulsory surgery.

[Preliminary results of treatment of solid tumors in children with non-standard radiotherapy]. / Predvaritel'nye rezul'taty lecheniia solidnykh opukholei u detei s ispol'zovaniem netraditsionnogo rezhima luchevoi terapii.

The efficacy and toxicity-related results of non-standard irradiation used in the combined treatment of 129 children with solid tumors were evaluated in 1986-1993. Immediate response (complete or partial regression) was registered in 89.3% of cases of sarcoma of Ewing, 76.8%--rhabdomyosarcoma and 76.7%--other tumors. Local recurrence was observed in 4 and late-onset complication of radiation injury in 10 patients. The actuarial 5-year survival was 54.3, 52.1 and 41.2% in Ewing sarcoma, rhabdomyosarcoma and other tumor groups, respectively.

[Proteolytic enzymes from Streptomyces fradiae: a metalloendopeptidase, subtilisin-like, and trypsin-like proteinases]. / Proteoliticheskie fermenty Streptomyces fradiae: metalloéndopeptidaza, subtilizinopodobnaia i tripsinopodobnaia proteinazy.

Three proteolytic enzymes-the metalloproteinase, SFMP, and two serine proteinases, SFSP and SFTP-have been isolated and purified from the culture fluid of Streptomyces fradiae using chromatography on bacitracin-silochrome, bacitracin-Sepharose, DEAE-cellulose and fractionation by ammonium sulfate. Study of physico-chemical and functional properties of the enzymes and structural analysis revealed that SFMP is a cysteine-containing metalloendopeptidase with M(r) of 36 kDa, has a peak activity for synthetic substrates at pH 7.0-7.5 and at 60-65 degrees C and is stable at pH 7.0-9.0. The serine proteinase SFSP is related to subtilisin-like enzymes, has a M(r) of 29 kDa and a pH optimum at 7.5-8.5 at temperature up to 50 degrees C. The proteinase is stable at pH 4.0-9.0 and retains 30% of its activity at 70 degrees C. The other serine proteinase, SFTP, has a M(r) of 26 kDa and is related to trypsin-like enzymes. Its activity for synthetic substrates of trypsin is maximal at pH 6.8-8.8 at 50 degrees C. The enzyme is stable at pH 4.5-8.5 and at temperature below 50 degrees C. It has been shown that Streptomyces fradiae, like Streptomyces griseus and other Streptomycetes, possesses an ability to secrete serine proteinases (SFSP and SFTP) related to two evolutionally distinct families of serine proteinases, i.e., subtilisin and chymotrypsin families. SFMP and SFSP have been isolated and characterized for the first time.

Physiology and genetics of antibiotic production and resistance.

Actinomycetes have the genetic capability to synthesize many different biologically active secondary metabolites and of these compounds, antibiotics predominate in therapeutic and commercial importance. Intensive research often centres on the use of molecular techniques to investigate the physiology and genetics of antibiotic biosynthesis with a view to improving production. The isolation of clones of Streptomyces hygroscopicus, the producer of geldanamycin, which synthesizes geldanamycin in S. lividans, is reported. Molecular approaches using genes for elongation factors (tuf) were used in attempts to increase the fermentation yield of kirromycin, whilst probes for aphD and sph, genes for streptomycin phosphotransferases, were used to gather information on streptomycin genes in soil. Actinomycete populations in soil and earthworms may help in developing a strategy for discovering additional antimicrobials in soil. The relationship of proline metabolism to the secondary metabolite undecylprodigiosin and the carbon regulation of spiramycin biosynthesis in S. ambofaciens is also reported.

[Isolation and characteristics of Bacillus megaterium metalloproteinase]. / Vydelenie i kharakteristika metalloproteinazy Bacillus megaterium.

Stepwise application of affinity chromatography on bacitracin-silochrome, gel filtration on Acrylex P-10, rechromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-15, a homogeneous metalloproteinase (M(r) = 35,000 Da) has been isolated from the cultural filtrate of B. megaterium strain 599. The amino acid composition and N-terminal sequence (20 amino acids) of the enzyme have been determined. The proteinase is not inhibited by diisopropyl-fluorophosphate, is inhibited by o-phenanthroline, EDTA, and Zn2+, and is activated by Co2+. The enzyme has a peak activity at 60-65 degrees C. The maximum of the enzymatic activity after hydrolysis of synthetic substrates is at pH 6.5-7.0. The enzyme is stable at pH 7.0-9.0 and retains its stability at 45-60 C for several hours. In acid media the enzyme undergoes irreversible inactivation. The dependence of kcat/Km on pH points to the involvement of an ionogenic group with pKa 7.5 in the catalytic act, most probably of the imidazole group of histidine. The metalloproteinase hydrolyzes synthetic peptide substrates at the bonds formed by the amino groups of hydrophobic amino acids-Phe, Leu, Ile and Val.

[Isolation and properties of serine proteinase from Aspergillus oryzae]. / Vydelenie i svoistva serinovoi proteinazy aspergillus oryzae.

A serine proteinase having an activity optimum at pH 6.7-8.2 has been isolated from amylorisine P-10x (a mixture of Aspergillus oryzae enzymes) by chromatography on DEAE-Sephadex A-50 and bacitracin Sepharose 4B. The proteinase is fully inactivated by phenylmethylsulfonylfluoride and diisopropylfluorophosphonate, the specific inhibitors of the enzyme, and has a pI at pH 7.5. The molecular mass of serine proteinase is 30000 Da; its amino acid composition appears as: Met2, Asp33, Thr18, Ser29, Glu21, Pro9, Glu32, Ala38, Val24, Ile16, Leu15, Tyr8, Phe8, His8, Lys18, Arg4, Trp6. The N-terminal sequence of the serine proteinase: Gly-Leu-Thr-Thr-Gln-Lys-Ser-Ala-Pro-Trp-Gly-Leu-Gly-Ser-Ile-Ser-Xaa-Lys- Gly-Gln-Gln-Ser-Thr-Asp-Tyr-Ile-Tyr, which coincides practically completely with the corresponding sequence of alkaline proteinase of A. oryzae, ATCC20386, has been determined. Similar to subtilisin, the enzyme catalyzes the condensation of leucine and alanine p-nitroanilides with N-benzyloxycarbonyl-alanyl-alanine and glycyl-alanine methyl esters.