Sequence-selectivity of 5,11-dimethyl-5H-indolo[2,3-b]quinoline binding to DNA. Footprinting and molecular modeling studies

Indolo[2,3-b]quinolines are a new family of the DNA intercalators showing significant cytotoxic activity. The mechanism of their action is based on the inhibition of DNA topoisomerase II activity. It depends on their ability to induce and stabilize drug-topII-DNA cleavable complexes. Site-specific intercalation of 5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ) was analyzed in vitro by DNaseI footprinting and by molecular modeling. To model the DNA-intercalator complex, use was made of the CVFF and ESFF force fields implemented in Insight 97.0 software. Experimental results were verified using a simple statistical model. The DiMIQ molecule was found to bind preferentially to the pBR322 DNA plasmid in the 5'-TGCTAACGC-3' region between adjacent adenine bases.     

Expression of the Arabidopsis thaliana AtJ2 cochaperone protein in Pichia pastoris

A vector was constructed for intracellular expression of the Arabidopsis thaliana DnaJ homologue AtJ2 in the methylotrophic yeast Pichia pastoris. The vector includes DNA encoding an amino-terminal histidine-tag, to simplify protein purification. Shake-flask cultures could be induced to produce approximately 250 mg/ L of AtJ2. Purified recombinant AtJ2 was able to stimulate the ATPase activities of both the Escherichia coli and Zea mays cytoplasmic Stress70 chaperone proteins five- to ninefold. The carboxy terminus of AtJ2 is -CAQQ, a protein farnesylation motif. When transformed P. pastoris was induced to synthesize AtJ2 in the presence of [(3)H]mevalonolactone, radioactivity was incorporated into the protein, suggesting farnesylation.     

Clonal Analysis Provides Evidence for Transient Initial Cells in Shoot Apical Meristems of Seed Plants

Abstract Drift of mutated sectors in sectorial or mericlinal plant chimeras has been interpreted as indirect evidence of initial impermanence at the apex. However, the same effect may result from mutation in noninitial cells positioned close to the vertex of the apical dome. Clonal analysis of the cell packets present in the superficial layer of spruce and magnolia apices provided the library of patterns suggesting that the position and the number of initial cells, and in some cases also the meristem axis inclination, may change over time. Multicellular clones originating from a single cell have been found in the geometric center of some apices, whereas in other apices the cellular center (where three or four clonal borders meet) did not correspond to the geometric center of the apex. Such effects may result only from initial impermanence.     

Delay inhibition of the active avoidance reflex and intertrial responses in rats in the shuttle box

A diminished probability of avoidance response in early phases of a warning signal was revealed with salient signals given after short intertrial intervals. The inhibition of the delay in avoidance response is due to an interaction of the safety state conditioning and the excitation elicited by onset of warning signal.     

Structure of the O-specific polysaccharide of Mesorhizobium huakuii IFO15243T

The structure of the O-specific polysaccharide isolated by mild acid hydrolysis of the lipopolysaccharide of Mesorhizobium huakuii IFO15243T was studied using methylation analysis and various one- and two-dimensional 1H and 13C NMR experiments. The O-antigen polysaccharide was found to be linear polymer constituted by a trisaccharide repeating unit of the following structure: --> 2)-alpha-L-6dTalp-(1 --> 3)-alpha-L-6dTalp-(1 --> 2)-alpha-L-Rhap-(1 -->.     

Adhesion of blood platelets to collagen and fibrinogen after treatment with cisplatin and its complex with glutathione

Cisplatin (cis-diamminedichloroplatinum II, CDDP) is one of the most widely used chemotherapy drugs. Unfortunately, it induces serious side effects such as haematological toxicity. The aim of the present study was to evaluate the effect of CDDP on the first step in blood platelet activation-platelet adhesion, induced by thrombin or adenosine diphosphate (ADP), to collagen and fibrinogen. The action of cisplatin was compared with the action of cisplatin glutathione complex (GS-Pt) on platelet adhesion and on free radical generation measured by chemiluminescence. Pretreatment of blood platelets with cisplatin (0.1-20 microM) caused a dose- and time-dependent reduction of platelet adhesion to collagen and fibrinogen (p <0.05). The GS-Pt complex (20 microM, 30 min) had a stronger inhibitory effect on this process. Moreover, the complex (R2 = 0.992; p <0.05) also stimulated the chemiluminescence of blood platelets to a greater extent than CDDP alone (R2 = 0.999; p <0.01). The results suggest that inhibition of platelet adhesion in the presence of cisplatin and its complex with glutathione correlates with the generation of reactive oxygen species in these cells.     

Some oncogene and tumour suppressor gene protein products expression in B-cell chronic lymphocytic leukaemia

The expression of Bcl-2, P53 proteins and known markers of proliferation, namely proliferating cell nuclear antigen (PCNA) and Ki67, in 29 patients with B-cell chronic lymphocytic leukaemia (B-CLL) was investigated. All leukaemic patients were classified, and immunophenotyped by the two-colour immunofluorescence method with the use of fluorocytometry. B-CLL was heterogeneous in the range of biological parameters of tumour cells. B-CLL patients manifested 34% positive Ki67 and 61% PCNA expression, whereas Bcl-2 and P53 positivity was 81% and 42%, respectively. The level of intracellular expression of Bcl-2 and P53 proteins did not depend on the stage of disease estimated by routine methods. Ki67 and PCNA expression was significantly higher in B-CLL patients with more advanced stages of the disease. A statistically significant correlation was established between their mutual expression.     

The synthesis of diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside hydrochloride

The N-trifluoroacetyl- and N-tetrachlorophthaloyl-protected bromide of D-glucosamine has been used for the first time as a glycosyl donor for the glycosylation of diosgenin [(25R)-spirost-5-en-3beta-ol]. Both 1,3,4,6-tetra-O-acetyl-2-deoxy-2-trifluoroacetamido-beta-D-glucopy ranoside and 1,3,4,6-tetra-O-acetyl-2-deoxy-2-tetrachlorophthalimido-alpha,beta -D-glucopyranoside were transformed into the appropriate glycosyl bromides. These reacted with diosgenin under mild conditions, using silver triflate as a promoter, and gave the corresponding protected diosgenyl glycosides. Each was deprotected to give diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside hydrochloride. The structures of the new glycosides were established by 1H NMR spectroscopy.