A new endogenous differentiating factor (myelopeptide-4) for myeloid cells.

Along with known lymphokines involved in the regulation of hematopoiesis, a new differentiating factor (myelopeptide-4, MP-4) for myeloid cells was found. The peptide (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro) originally isolated from the culture medium of porcine bone marrow cell culture was examined for its ability to induce differentiation in two human myeloid leukemia cell lines, HL-60 and K-562. Agents with well-known differentiation-inducing activity, such as phorbol myristate acetate, dimethylsulfoxide and the lymphokines were used as a reference. It has been shown that MP-4 significantly influences the integral characteristics of metabolism, expression of surface antigens and morphology of these cells. It decreased the level of chromosomal DNA synthesis and, in parallel, increased the total protein synthesis in both HL-60 and K-562 cells. MP-4 induced the expression of CD14 monocyte-specific surface antigen and the appearance of mature monocytes/macrophages in HL-60 cell cultures. There was a good correlation of cell metabolic/morphological changes and the CD14 marker expression for HL-60 cells. A similar phenomenon was observed in K-562 cells treated with MP-4 when the levels of hemoglobin synthesis were detected in their cytoplasm. Thus, we consider MP-4 as a new endogenous differentiating factor for myeloid cells.

The bone marrow peptide (myelopeptide-2) abolishes induced by human leukemia HL-60 cell suppression of T lymphocytes.

Myelopeptide-2 (MP-2) Leu-Val-Val-Tyr-Pro-Trp originally isolated from the supernatant of porcine bone marrow cell culture was examined for its capacity to restore the mitogen responsiveness of human T lymphocytes inhibited by conditioned media from HL-60 leukemia cells (HL-60 CM). MP-2 added to phytohemagglutinin (PHA)-stimulated T lymphocytes together with HL-60 CM abolished the suppression of T-lymphocyte proliferative response in a dose-dependent manner. Another bone marrow hexapeptide Phe-Leu-Gly-Phe-Pro-Thr, MP-1, did not display this action in that experimental system. MP-2 was also effective being added after T-lymphocyte exposure to HL-60 CM which suggests its recovery but not protective effect on T-lymphocytes treated with tumor cell products. Flow cytometry analysis revealed HL-60 CM influence on the expression of CD3 and CD4 T-cell surface antigens. It decreased the content of CD3- and CD4-positive cells and induced the appearance of T lymphocytes with reduced density of CD3 and CD4 antigens. MP-2 was able to restore the T-cell phenotype altered by HL-60 CM. MP-2 seems to be promising in anti-tumor therapy.

[The role for nucleus-associated polyribosomes in the coordination of histone synthesis with replication of chromosomal DNA in loach embryonic cells]. / Rol' iadernosviazannykh poliribosom v koordinatsii sinteza gistonov s replikatsiei khromosomnoi DNK v émbrional'nykh kletkakh v'iuna.

During intensive chromosome replication in loach (Misgurnus fossilis) embryonic cells a particular class of nucleus-associated polyribosomes has been discovered alongside the free and membranebound polyribosomes. The comparison of the translational patterns of these three polyribosome classes in in vivo and in vitro conditions has proved their metabolic and functional difference. It has been shown that the nucleus-associated polyribosomes comprising over 50 per cent of all the cell polyribosomes in the above-mentioned period produce the bulk of lysine-rich histones on the newly formed mRNAs. Using metabolic inhibitors of DNA, or RNA, or protein synthesis it has been established that the nucleus-associated polyribosome functional activity as differs from that of free and membrane-bound polyribosomes and depends to great degree on the chromosomal DNA replication and the shortliving mRNAs transcription. The data obtained suggest that the chromatin biogenesis control can be achieved by spacial coupling between DNA replication and histone synthesis at the nuclear envelope level.

[Transcription of ribosomal genes in nuclei of different ploidy]. / Transkriptsiia ribosomnykh genov v zarodyshakh razlicnoi ploidnosti

It has been shown previously that the production of "mature" (27S and 18S) ribosomal RNA's (rRNA) at late stages of embryonic development of the loach (Misgurnus fossilis) does not depend on the degree of ploidy, the haploid (1n) embryos making, per cell, as much rRNA as do the diploid (2n) ones. In order to investigate the mechanims of the compensation of rRNA production in 1n embryos, incorporation of labelled nucleosides in different RNA fractions was compared in two genetical variants. The kinetic and sedimentation analysis showed that in 1n cells a compensatory increase in the production of 36S precursor of rRNA (pre-rRNA) takes place. Chase experiments showed that the rate of the processing and decay of the pre-rRNA was similar in the two genetic forms. Saturation hybridization of labelled, purified 27S rRNA with DNA isolated from the nuclei of 1n and 2n embryos failed to reveal any significant differential increase (amplification) in the proportion of ribosomal cistrons (rDNA) in the haploid genome. It is concluded that the compensation of the production of rRNA in the haploid embryos is accounted for by a greater rate of transcription of the totality of the ribosomal cistrons per single chromosome set.

[Ribosomal RNA synthesis in haploid and diploid loach embryos]. / Sintez ribosomnykh RNK v gaploidnykh i diploidnykh embrionakh v'iuna

rRNA synthesis was compared in the loach haploid (In) and diploid (2n) embryos. The relative intensity of synthesis was evaluated by 14C-uridine incorporation in 27S and 18S rRNA isolated from ribosomes taking into account label incorporation into total acid-soluble fraction and phosphrylated uridine derivatives. Label incorporation into rRNA, in reference with DNA content in 1n and 2n embryos, suggests that the level of rRNA synthesis per DNA unit in haploids is twice that in diploids whereas, in reference per cell, the same amount of ribosomes is synthesized both in haploids and diploids. The data obtained show that the amount of rRNAs synthesized in the loach embryogenesis does not depend on ploidy.

[Kinetics of the synthesis of nucleic acids in haploid and diploid loach embryos]. / Kinetika sinteza nukleinovykh kislot v gaploidnykh i diploidnykh émbrionakh v'iuna

Al kinetic analysis of incorporation of the mixture of 3H-nucleosides in the nucleic acid fractions was carried out to examine the mechanisms of compensation of the genetic material deficiency. Both the haploid and diploid embryos of the loach (Misgurnus fossilis L.) were analyzed. When comparing the DNA and RNA syntheses, the level of phosphorylation (nucleotide pool) in the both genetic variants was under control. The rate of incorporation of the labelled nucleosides in DNA was shown to be higher in haploids at the early developmental stages than in diploids but later it became the same. The increased level of DNA replication in the early haploid embryos was due to the compensatory increase of the cell number in them as compared with the diploid ones. The rate of total RNA synthesis corrected by the differences in the rate of nucleoside phosphorylation varied directly with the degree of ploidy at the blastula stage; at the gastrula stage the value of RNA synthesis per haploid genome was compensated, and at the stage of organogenesis the production of total RNA, as calculated per cell, became in haploids even higher than in diploids. The data obtained suggest the essential changes in the patterns of RNA synthesis control during development.

[Early activation of ribosomal RNA synthesis in axolotl embryos]. / Ranniaia aktivatsiia sinteza ribosomnykh RNK u zarodyshei aksolotlia.

The incorporation of labeled precursors (3H- and 14C-uridine) into the fractions of salt-insoluble RNAs and or rRNA was studied in the axolotl embryos at different stages of early development (from the end of synchronous cleavage divisions until the end of gastrulation). RNA preparations isolated from the embryos at all stages studied contained incorporated radioactivity. The radioactivity of salt-insoluble RNAs markedly increased at the beginning of blastulation and continued to grow, but less steeply, at the subsequent developmental stages. The sedimentation analysis has shown that the label is incorporated into the precursors and mature molecules of rRNA, beginning at least from the blastulation.

[DNA and the theory of stabilizing selection]. / DNK i teoriia stabiliziruiushchego otbora.

A comparison of structural-functional features of genomic DNAs allowed to estimate the role of internal and external factors in evolution of different groups of organisms. The basic difference between higher and lower organisms has been demonstrated. It is reflected in the difference of their reaction on to external factors in accordance with two adaptation types, the openness and autonomization. There is a correlation between structural-functional organization of genomic DNAs of higher and lower organisms and the above mentioned types of adaptation. DNA of lower organisms has been proposed to be characterized as "labile", and that of higher organisms, as "stable". The "DNA lability" means high mutation ability, which characterizes the existence of and evolution of lower organisms (genetic inconstancy of the lower organisms). On the contrary, "DNA stability" means the creation of stable genetic apparatus, reduction of variability in higher organisms (genetic constancy of higher organisms). This suggests the existence of the two principal ways of evolution.

[Properties of polyribosomal complexes of loach embryonic cell cytoplasm]. / Kharakteristika poliribosomnykh kompleksov tsitoplazmy embrional'nykh kletok v'iuna.

The translation-active complexes of the extranuclear region of cytoplasm of developing loach (Misgurnus fossilis) embryos were studied. The amount of ribosomes involved in polyribosomal complexes was shown to increase 6-fold from the blastula stage to organogenesis, making up only a small (3% and 18%, respectively) part of the total pool of cytoplasmic ribosomes. Free polyribosomes make up the bulk (up to 95%) of the total cytoplasmic polyribosomal pool at early stages of development (blastula, gastrula) and about 50% at late stages (organogenesis). The membrane-bound polyribosomes appear in early gastrulation; their content reaches its maximum (40-45%) in organogenesis. The newly formed RNAs liberated from the nucleus into the extranuclear space of the cytoplasm in the course of ontogenesis are incorporated into the polyribosomes (predominantly membrane-bound ones) not before the gastrulation starts. The proportion of these poly(A)-enriched mRNAs in the polyribosomes does not exceed 10% of the total amount of newly synthesized cytoplasmic RNAs at this stage. A population of light polyribosomes capable to synthesize histones was detected in free polyribosomes, which do not practically contain the newly formed RNAs. The data obtained are discussed in terms of the literary data on the type of RNA and protein synthesis in early ontogenesis.

[Effect of myelopeptides on the synthesis of DNA and total protein in the cells of the lymphoid organs in the mouse]. / Izuchenie deistviia mielopeptidov na sintez DNK i summarnogo belka v kletkakh limfoidnykh organov myshi

Using double 3H-thymidine/14C-amino acid label, the influence of myelopeptides (MP) on chromosomal DNA and total protein (without histones) synthesis has been studied in vitro in mouse lymphoid organ cells. It has been shown that MP cause a decrease in DNA labelling and a parallel increase in protein labelling in bone marrow cells and have practically no effect on these two parameters in thymus, lymph node and spleen cells. It has been found that MP have only mitogenic effect on the above-mentioned organ cells, but in combination with 2-mercaptoethanol. The data obtained show that MP possess the properties of cell-differentiating factors.

[Changes in the representation of repeating DNA sequences in nuclear RNAs of early loach embryos]. / Izmeneniia predstavlennosti povtoriaiushchikhsia posledovatel'nostei DNK v iadernykh RNK rannikh émbrionov v'iuna.

The hybridization kinetics of nuclear RNAs of loach embryos labelled with [3H]uridine for 1 hour with DNA excess shows that during embryogenesis (from the blastula to the gastrula stage) the number of newly formed RNA molecules transcribed from repeating DNA sequences in considerably reduced. This occurs both in the RNA fraction extracted from the nuclei with phenol pH 7.7 and having a low sedimentation coefficient and a low degree of polyadenylation, and in the RNA fraction extracted with phenol pH 9.0 having a higher sedimentation coefficient and a higher degree of polyadenylation.

Multiple anchoring of myelopeptides on sequential oligopeptide carriers (SOC(n)): synthesis, conformation and studies in human leukemia cells.

Myelopeptides, MP-6 (Val-Asp-Pro-Pro) and MP-4 (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro), induce metabolic changes in human leukemia cells, HL-60, characteristic of the differentiation process, which should be regarded as a promising therapeutic approach in cancer and related diseases. With the aim to optimize the differentiation effect of MPs, they were coupled to the Lys-N(epsilon)H(2) groups of a sequential oligopeptide carrier Ac-(Lys-Aib-Gly)(4), SOC(4), and the constructs obtained were studied. The rigid 3(10) secondary structure of the carrier is preserved even after linkage of the MPs, which also maintain their initial conformations without interacting either with each other or with the carrier, as demonstrated by (1)H nuclear magnetic resonance (NMR) spectroscopy. It is concluded that the carrier accommodates the presentation of MPs, thus improving their differentiation effect on human leukemia cells.