Synthesis and antitumor activity of betulin, erythrodiol and uvaol aminopropoxy derivatives

The synthesis of aminopropoxy derivatives of betulin, erythrodiol, uvaol and oleantriol via cyanoethylation of triterpenoids hydroxyl groups and subsequent reduction of cyanoethyl fragments is described. High and specific in vitro antitumor activity (cytotoxicity) of 3beta,28-di-O-[3-(aminopropoxy)]lupa-20(29)-ene and 3beta-O-hydroxy-28-O-[3-(aminopropoxy)]olean-12-ene towards a wide range of human tumor cell lines is discovered. The aminopropoxy group is shown to be a new perspective pharmacophor group for design of anticancer agents on the basis of triterpenoids.     

Proton magnetic resonance spectroscopy in the diagnosis of breast cancer

Magnetic resonance spectroscopy (MRS) is used in mammalogy to specify the data obtained by traditional radiodiagnostic techniques and provides a possibility of estimating the molecular composition of pathologically altered breast tissues. This information is employed for the differential diagnosis of malignant and benign neoplasms, as well as for monitoring and predicting the efficiency of chemotherapy for breast cancer. Some technical problems and the lack of a standardized approach to the procedure of a study and interpretation of its results hinder the wide introduction of spectroscopy into clinical practice. This paper reviews the data available in the literature on proton MRS and the technical and clinical aspects of application of this technique.     

Analysis of glycogen structure in rat hepatocytes using cytochemical and FRET methods

Using cytochemical and FRET (Forster, Resonance Energy Transfer) methods, the glycogen structure in rat hepatocytes was investigated during fasting and at different time intervals after per os glucose administration to animals. Hepatocytes on slides were stained with fluorescent PAS-reaction. Staining the slides with ethidium bromide-SO2 (EtBr-SO2) for 40 min revealed a labile glycogen fraction (LE), and the subsequent staining the same samples with auramine-SO2 (Au-SO2) for 50 min showed a stable glycogen fraction (SF) in the cells. The total glycogen content (LF and SF) in the hepatocytes at different stages of refeeding was determined by means of cytofluorimetry, and then efficiency of FRET was measured in the same cells. Registration of FRET in several areas of the cells was carried out on a laser scanning confocal microscope Leica TCS SP5 with application of FRET AB (Acceptor Photobleaching) procedure. In this procedure, auramine served as a donor (D) and ethidium bromide was an acceptor (A). It was shown that the efficiency of FRET varied from 10 to 14 % during refeeding, while the glycogen structure had a marked influence on the value of this parameter. FRET efficiency was shown to correlate with the ratio A/D in the cells of hungry rats and at the early stages after glucose administration to animals, which reflected the degree of filling of the external tiers of glycogen molecules of glucose residues. At later stages, this correlation was either less pronounced or absent. It was found that the FRET efficiency can vary by 3-4 times at the same value of A/D. Since the probability of energy transfer from D to A is proportional to 1/R6, where R is a distance between D and A, such variations of the FRET efficiency indicate that the glycogen molecules possess a labile structure in which the chain of glucose residues can deviate from its axis by a distance of about half their diameter.     

Clinical application of VNTR-typing of Mycobacteria tuberculosis strains: monitoring of treatment quality and of laboratory service

An early diagnosis of super-infection and mixed infection of Mycobacteria tuberculosis is highly important for the correction of an assigned treatment and for a positive prognostication. An analysis of loci with varying tandem repeats (VNTR) is a simple in use and reproducible method of M. tuberculosis genotyping. Thirty-seven serial isolates of M. tuberculosis from 12 patients with pulmonary tuberculosis including changing resistance to TB drugs registered in follow-up and treatment underwent VNTR-typing for 6 loci: ETR-A, C, E, V1, V3 and V4. Both super-infection and mixed infection were shown in 33.3% of cases to cause changes in the profile of resistance to TB drugs. In 3 patients, changes in the drug resistance profile were accompanied by a substitution of a colonizing strain for an epidemic clonal variant of the Beijing family with the 445446 genotype. Whereas in one patient, there was a substitution of the Beijing strain for the genotype 222422 strain, which is typical of M. tuberculosis with the S42 spoligotype. One intermediate serial isolate from the above patient had a mixed culture of 2 polyresistant strains of M. tuberculosis (445476 + 222422). In one case, the substitution of the infecting strain for a strain with changed genotypes and profiles of resistance to antibiotics occurred twice within an interval of 5-6 months. The 343543 genotype strain changed for the 452562 genotype strain and then--for the 445466 genotype strain; the final genotype variant belonged to the Beijing family. Serial isolates from 8 patients retained their original genotype (Beijing). In such cases, the changing spectrum of resistance to TB drugs can be associated with the secondary drug resistance acquired by strains in the process of treatment or with errors of laboratory equipment. Finally, the VNTR analysis is a rapid, easy-in-use and effective tool for the systemic typing of M. tuberculosis isolates in clinical practice.     

Characteristics of some stages of energy metabolism and antioxidant system in bone marrow myeloid cells and leukocytes from piglets

The age changes of enzymes of activity catalyzing several links of energy metabolism (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase, cytochrome c-oxidase) and antioxidant system (superoxide dismutase and glutathione reductase) in bone marrow myeloid cells and blood leukocytes of pig in the 10-day period after birth were investigated. The bone marrow cells and leukocytes of the new born piglets were characterized by low intensity of oxidative steps of energy metabolism as well as by low activity of antioxidant enzymes. In the period of neonatal adaptation reorganization of energy metabolism, particularly, intensification of oxidative processes in the investigated cells occurred. It included the pentose phosphate way and cytochrome c-oxidase activation. During the neonatal period of development the functional activity of antioxidant enzymes in the investigated cells of piglets increased.     

Point amino acid substitutions in the Ca2+-binding centers of recoverin. I. Mechanism of successive filling of Ca2+-binding centers

The molecule of photoreceptor Ca(2+)-binding protein recoverin contains four potential Ca(2+)-binding sites of the EF-hand type, but only two of them (the second and the third) can actually bind calcium ions. We studied the interaction of Ca2+ with recoverin and its mutant forms containing point amino acid substitutions at the working Ca(2+)-binding sites by measuring the intrinsic protein fluorescence and found that the substitution of Gln for Glu residues chelating Ca2+ in one (the second or the third) or simultaneously in both (the second and the third) Ca(2+)-binding sites changes the affinity of the protein to Ca2+ ions in different ways. The Gln for Glu121 substitution in the third site and the simultaneous Gln substitutions in the second (for Glu85) and in the third (for Glu121) sites result in the complete loss of the capability of recoverin for a strong binding of Ca(2+)-ions. On the other hand, the Gln for Glu85 substitution only in the second site moderately affects its affinity to the cation. Hence, we assumed that recoverin successively binds Ca(2+)-ions: the second site is filled with the cation only after the third site has been filled. The binding constants for the third and the second Ca(2+)-binding sites of recoverin determined by spectrofluorimetric titration are 3.7 x 10(6) and 3.1 x 10(5) M-1, respectively.