[Pharmacodynamics, safety, and clinical effects of a novel glycoprotein IIb/IIIa antagonist framon during high risk coronary angioplasty]. / Farmakodinamika, bezopasnost' i klinicheskie éffekty novogo antagonista glikoproteidov IIb/IIIa framona pri koronarnoi angioplastike vysokogo riska.

Preparation framon [F(ab')2 fragments of the anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (FRaMon)] blocks fibrinogen binding to GP IIb/IIIa and platelet aggregation. Dynamics of platelet aggregation inhibition, safety, and clinical effects of framon were studied in high-risk coronary angioplasty. Twenty seven patients underwent angioplasty with framon, 29 - with abciximab and 28 - with no GP IIb/IIIa antagonists. Framon at 0.2 mg/kg (n=16) and 0.25 mg/kg (n=11) bolus administration inhibited platelet aggregation induced by 20 mcM ADP by more than 90%, 80%, 60% and 30% in comparison with the predrug level 1, 12, 24 and 72 h after injection, respectively. Almost the same dynamics of aggregation inhibition was observed upon abciximab administration at 0.25 mg/kg bolus + 0.125 mcg/kg/min infusion for 12 h. No signs of individual intolerance and side effects including allergic reactions and bleedings were detected in patients treated with framon. Slight decrease of platelet count (15-20%) was observed on the first day after framon administration. Antibodies against framon were detected in 1 out of 22 tested patients. Free (nonbound to platelets) framon was completely removed from the circulation 12 h after injection. The number of endpoints (death, myocardial infarction and indications for repeat revascularization) within 1 year after angioplasty was approximately the same in the groups with framon and abciximab - 7 of 25 (28%) and 7 of 28 (25%), respectively, and more than 1.5 fold higher in the group without GP IIb/IIIa blockers - 12 of 27 (44,4%).

A major nucleolar protein B23 as a marker of proliferation activity of human peripheral lymphocytes.

A novel monoclonal antibody (Mab) (called 3C9) against a major nucleolar phosphoprotein B23 was used to study B23 qualitative and quantitative alterations in phytohemagglutinin (PHA) -stimulated human peripheral blood lymphocytes in indirect immunofluorescence and Western blots. It was shown that lymphocyte proliferation was accompanied by gradual augmentation of nucleoli and their accumulation of the protein B23 up to 2-fold by 16 h and 40-50 fold by 72 h, as compared with the non-stimulated cells. By parallel immunolabeling with the anti-Ki-67 antibody, it was shown that the early changes of B23 amount and localization occurred before an appearance of Ki-67 protein, a well-known marker of proliferating cells. Our results evidence that antibodies against B23 might be applied for recognition of human peripheral lymphocytes at early stages of their activation for proliferation, preceding the S-phase.

[Characteristics of nucleolar organization and function under condition of complete spatial in situ disassembly of its main structural components]. / Osobennosti organizatsii i funktsionirovaniia iadryshka v usloviiakh polnogo prostranstvennogo razobshcheniia ego osnovnykh strukturnykh komponentov in situ.

It is well known that fibrillar centers (FC) constitute an essential structural component of the active nucleolus in mammalian cells, yet their role in regulation of ribosomal gene transcription still remains an open question. Here, we studied the activity of endogenous RNA polymerase I upon partial and complete unraveling of nucleoli and FCs. The pattern of BrUTP incorporation in nuclei of hypotonically-treated cells was shown to be essentially the same as in the control untreated cells. Moreover, the sites of BrUTP incorporation, which revealed the active PNA polymerase I, were completely coincident with UBF-binding sites. These observations allow to conclude that structural integrity of FCs is not a prerequisite for maintenance of the active RNA polymerase I transcriptional complex. When the action of hypotonic shock was ceased and the cells were transferred to a complete cultural medium, the swollen nucleoli recovered to the control state. Therefore it is possible to conclude that none of the main morphological nucleolar counterparts, such as FCs, dense fibrillar component or the pars granulosa, is responsible for the maintenance of the nucleolar structural and functional integrity. A suggestion is made that this role may be played by the nucleolar matrix associated with the RNA polymerase I transcriptional complex.

[Inhibition of thrombocyte aggregation by F(ab')2-fragments of monoclonal antibodies FraMon (CRC64) to glycoproteins IIb-IIIa]. / Ingibirovanie aggregatsii trombotsitov F(ab')2-fragmentami monoklonal'nogo antitela FraMon (CRC64) k glikoproteidam IIb-IIIa.

AIM: To study the effects of F(ab')2 fragments of the monoclonal antibody (monAB) FRaMon against glycoproteins (GP) IIb-IIIa on platelet aggregating activity in vitro and after injection to healthy volunteers. MATERIAL AND METHODS: In vitro experiments were performed to study effects of F(ab')2 and Fab fragments of FRaMon on platelet aggregation (PA) and 14C-serotonin secretion and characteristics of 125I-labelled F(ab')2 and Fab FRaMon binding to platelets. In vivo effects of F(ab')2 FRaMon (preparation FRAMON) were studied upon its bolus i.v. injection to 10 healthy volunteers at the doses of 0.025-0.2 mg/kg. PA was registered before and 1, 6, 12, 24 hours and 3 and 12-15 days after FRAMON injection. FRAMON binding to platelets in the vascular bed was evaluated by inhibition of 125I-FRAMON in vitro binding to platelets obtained from volunteers. Development of antibodies against FRAMON was evaluated by ELISA two weeks after FRAMON injection. RESULTS: In vitro F(ab')2 FRaMon completely blocked PA induced by ADP and thrombin at the concentrations < 4 and < 7.5 mcg/ml, respectively, and revealed higher inhibitory capacity than Fab FRaMon. F(ab')2 FRamon also inhibited 14C-serotonin secretion from ADP-activated platelets. F(ab')2 FRamon interacted with two GP IIb-IIIa molecules on one platelet and bound to platelets more tightly than Fab FRaMon. F(ab')2 FRaMon (preparation FRAMON) bolus i.v. injection to healthy volunteers at the doses of 0.025-0.2 mg/kg did not induce any signs of individual intolerance, including allergic reactions, bleeding and thrombocytopenia. FRAMON at 0.2 mg/kg almost completely inhibited ADP-induced PA of volunteer's platelets 1 h after injection and by more than 70% at 12 h and by more than 50% at 24 h after injection. PA ability recovered to normal 3 days after injection. Antibodies against FRAMON were detected in 1 out of 10 volunteers. CONCLUSION: F(ab')2 fragments of monAB FRaMon effectively inhibited aggregating ability both in vitro and after injection to healthy volunteers and could be suggested as a basis for development of a new GP IIb-IIIa antagonist.

[Identification of nucleolar antigen in different types of cells using domestic monoclonal antibodies]. / Vyiavlenie iadryshkogo antigena v razlichnykh tipakh kletok s pomoshch'iu otechestvennykh monoklonal'nykh antitel.

Murine hybridoma 3C9 producing BCA-PC monoclonal antibodies and selectively staining the nucleoli in various cells, most intensely in actively proliferating cell cultures, was obtained by somatic hybridization of cells. BCA-PC MAb belong to IgM. As a rule, IgM antibodies are virtually unavailable for intracellular antigens, and therefore a special method has been developed for fixation and performance of indirect immunofluorescence with BCA-PC. By its molecular weight (37 and 43 kD) and distribution in the cellular nucleus, the antigen detected by BCA-PC resembles the nucleolar protein B23 located in the nucleolar granules. The content of B23 increases with malignant degeneration of cells and during cell proliferation. It is therefore believed that the new MAbs will help characterize the proliferative status of cells which determines the malignancy of a pathological process.

[Analysis of the proliferative activity of a cell using new monoclonal antibodies to nucleolar protein B23/nucleophosmin]. / Analiz proliferativnoi aktivnosti kletok s pomoshch'iu novykh monoklonal';nykh antitel k iadryshkovomu belku B23/nukleofozminu.

Nowadays, antinucleolar antibodies are widely used for exploration of the nucleolar organization and molecular mechanisms of ribosome production. Here we have described a new monoclonal antibody against the major nucleolar phosphoprotein B23/nucleophosmin (3C9) that is involved in the terminal stages of ribosome production. It is used to examine immunocytochemical peculiarities of the nucleolus in terms of the cell proliferative status and also during mitosis. In human peripheral blood lymphocytes, activated for proliferation with phytohaemagglutinin (PHA), PHA stimulation of lymphocytes was shown to result in accumulation of protein B23 in augmentative nucleoli. A comparative study of 3C9 and two other anti-B23 antibodies 20B2 and anti-B23 by Western blots and indirect immunofluorescence favored the idea that 3C9 cross-reacted with the major isoform of B23, B23.1, that have an apparent molecular weight of 40 kDa.

[The dynamic intracellular distribution of nucleolar protein B23 in the interphase and mitotic cells of mammalian cultures]. / Dinamika vnutrikletochnogo raspredeleniia iadryshkovogo belka B23 v interfaznykh i mitoticheskikh kletkakh nekotorykh kul'tur mlekopitaiushchikh.

Using monoclonal antibodies against B23 (Ochs et al., 1983) and indirect immunofluorescence of cells exposed to different conditions of fixation, distribution of B23 in HeLa (human) PtK1 (rat kangaroo) and PK (pig) cells was studied at interphase and mitosis. In different cells B23 is distributed in a similar manner. At interphase it is located mainly within nucleoli. When cells enter mitosis (at prophase), B23 translocates from nucleoli to the nucleoplasm, and after breakdown of the nuclear envelope at prometaphase-to the cytoplasm to remain there till the end of telophase. As extraction buffers are applied to remove soluble cytoplasmic proteins. B23 is clearly seen on the surface of chromosomes. This unequivocally indicates that B23 is a "chromosomal passenger protein" (Earnshaw, Barnet, 1991). At telophase, the formation of numerous prenucleolar bodies on the surface of chromosomes starts. A long-term incubation of cells with actinomycin D prevents from the appearance of B23 around chromosomes and within prenucleolar bodies.

[The behavior of nucleolar proteins under the conditions of the reversible three-dimensional separation of the structural components of the nucleolus]. / Povedenie nekotorykh iadryshkovykh belkov v usloviiakh obratimogo prostranstvennogo razobshcheniia strukturnykh komponentov iadryshka.

The intracellular localization of Ag-proteins and protein B23 has been studied under the action of a low ionic strength solution (20% Hank's solution) on living pig cultured kidney cells (PK). For this, Ag-staining of nucleoli and indirect immunofluorescence using antibodies against B23 were applied. It has been shown that incubation of living cells in diluted Hank's solution results in migration of B23 from nucleoli to the nucleoplasm. A lot of Ag-proteins also migrate into the nucleoplasm, while part of them remained bound with the nucleolar remnants. Such a relocation of B23 was found to be completely reversible, and if hypotonic medium was replaced by the complete one, the nucleoli were able to reappear again, and B23 was detected solely within them. Fusion of prenucleolar bodies with reconstructing nucleoli could be delayed by cell incubation with actinomycin D. Generally, the process of nucleolar reconstitution following their artificial disorganization in hypotonic medium was believed to be similar to the nucleologenesis observed during normal mitosis. Light hypotonic treatment of living cells facilitates the evaluation of the number of silver grains over nucleoli due to their less compact arrangement than in the control.

[The effect of low ionic strength solutions on the structure and function of the nucleoli in living ESK cells]. / Vliianie rastvorov nizkoi ionnoi sily na struktury i funktsiiu iadryshek v zhivykh kletkakh SPEV.

The effect of a 20% Hanks [correction of Hanx] solution on living cells of pig embryo kidney culture induces sharp changes in the structural and functional organization of nucleoli. 10-15 min after the beginning of the effect, no nucleoli are observed under the light microscope. The ultrathin sections reveal only fibrillar centers (FC) and a dense fibrillar component. Under these conditions nucleoli do not incorporate 3H-uridine. However, 30-120 min after the cells are transferred from the hypotonic to the normal isotonic medium, the nucleoli cannot be distinguished from initial ones by their form, size, location in the nucleus, ultrastructure and the 3H-uridine labelling pattern. This indicates that the effect of a 20% Hanks [correction of Hanx] solution on nucleoli of living PE cells is reversible. It has been suggested that reconstruction of nucleoli in the full culture medium may proceed at the expense of both synthesis de novo and the material of initial nucleoli.