Synthetic insulin fragment with insulin-like biological activity.

An insulin fragment, representing the C-terminal functionally important site of its molecule and responsible for receptor binding, was synthesized. The fragment consists of two peptides: a dipeptide (A 20-21) and an octapeptide (B 19-26), linked with a disulfide bond (A20-B19). The biological activity of the newly synthesized fragment relative to insulin was assayed for the influence on glycogenesis and for the ability to stimulate glucose uptake. Comparative tests for the biological activity of the synthesized fragment and of the intact hormone allowed us to conclude that the fragment has insulin-like properties.

[Transfer of the functional genes into eucaryotic cells by neutral phospholipid liposomes]. / Perenos funktsional'nykh genov v kletki éukariot s pomoshch'iu neitral'nykh fosfolipidnykh liposom.

Transfection of plasmid DNAs containing b-galactosidase gene (pQE-LacZ) or alkaline phosphatase (pCSEAP) into L929 cell line using was studied. The complexes between plasmid DNA and liposomes containing Ca ions and glycyrrhizic acid or &-tocopherol caused successful transfection of functional genes into L929 cells. The efficiency of transfection of plasmid DNAs into L929 cells using polynucleotide-metallo(II)-liposome complexes were 30-50% from the efficiency value of calcium phosphate coprecipitation transfection.

[Nonviral methods of gene transfer in gene therapy]. / Nevirusnye metody perenosa genov v gennoi terapii.

A number of viral and non-viral vector systems have been developed nowadays for gene therapy applications. The advantages and shortcomings of the following non-viral methods of transfection are discussed in this review: calcium phosphate technique, ballistic transfection using "gene gun", electroporation, microinjection into the nucleus, receptor-mediated gene transfer, and artificial macromolecular complexes (polycations, hydrophobic polycations, polymers, cationic and neutral liposomes). Special attention is paid to methods of lipofection based on the usage of cationic and neutral liposomes as well as targeted gene delivery with the emphasis on the works which were out in author's laboratories.

[A synthetic fragment of insulin with insulin-like biological activity]. / Sinteticheskii fragment insulina s insulinopodobnoi biologicheskoi aktivnost'iu.

We have used computer modeling of insulin 3-D structure and experimental data about action of site point mutation on insulin activity to design functionally important domain with signaling activity and synthesized peptide than might be sufficient for the binding to insulin receptor. The designed and synthesized peptide consist of ten residues and may be obtained in two forms: oxidized and reduced (with or without disulfide bond). The synthesized decapeptide peptide represents functionally important site for binding to the insulin receptor. Amino acid residues at position 1-8 correlate with B-chain of insulin at position (B19-B26). Residues at position 9.10 correlate with A-chain at position A-10-A21. This peptide was tested with cell culture L-929 (glucose uptake) in comparison with bioactive commercial peptide (R-G-FF) and insulin. It was shown that synthesized peptide exhibit biological activity at molar concentration 0.01-1 mkM. Our results successfully demonstrate the synthetic insulin fragment have insulin-like biological activity.

[Stimulation of regenerative processes and correction of the functional activity of the liver in its partial resection and toxic lesions]. / Stimuliatsiia protsessov regeneratsii i korrektsiia funktsional'noi aktivnosti pecheni pri ee chastichnoi rezektsii i toksicheskikh porazheniiakh.

The effects of hepatotropic growth factors (HGFs) and phospholipid drugs on the recovery of functions and the regeneration of the rat liver were studied in CC14-induced toxic damage and after partial hepatectomy (PHE). HGFs isolated from the cytoplasmic cells of the regenerating liver, as well as from the liver of the animals given prodigiozan and from the media taken after culturing the explants of the regenerating liver were found to stimulate DNA synthesis and hepatocytic proliferation following PHE and in the cirrhotic liver. Prodigiozan was shown to induce the formation of HGFs not only in the rat liver following PHE, but in the liver of intact animals. It was established that the covalently binding complex of albumin and bilirubin stimulated the synthesis of proteins and DNA in the regenerating liver, but non-covalently binding complex inhibited these processes. When CC14 was administered to the animals, the two complexes enhanced the reparative synthesis of DNA, without changing the level of replicating synthesis, the non-covalently binding complex completely eliminating the single-strand breaks in DNA. Phospholipid agents containing soybean and sunflower phosphatidylcholines increased the synthesis of RNA and albumin, which were decreased due to exposure to CC14 and had the property of stimulating the synthesis of total DNA and considerably enhancing that of mitochondrial DNA.

[The effect of covalently and noncovalently bound complexes of albumin with bilirubin on DNA and protein synthesis in liver and spleen of rats subjected to splenectomy and partial hepatectomy]. / Vliianie kovalentno i nekovalentno sviazannykh kompleksov al'bumina s bilirubinom na sintez DNK i belka v pecheni i selezenke krys so splenéktomiei i chastichnoi gepatéktomiei.

Noncovalently bound complexes of albumin and bilirubin were found to stimulate DNA and protein synthesis in partially hepatectomized regenerating rat liver tissue, while the covalently bound complex inhibited both these synthesis types in liver tissue after partial hepatectomy. Splenectomy of intact rats caused an induction of DNA and protein synthesis in liver tissue but partial hepatectomy decreased drastically the synthesis rate in spleen, thus suggesting that humoral factors stimulating the proliferation response in liver and spleen tissues were developed after spleen- and hepatectomies. The covalently bound albumin and bilirubin complex did not affect the rate of DNA and protein synthesis in liver tissue of splenectomized rats, while the complex with noncovalent bonds restored the rate of DNA and protein synthesis in the spleen of rats with partial hepatectomy. Only the noncovalently bound complex of albumin and bilirubin exhibited the properties inherent in hepatotropic growing factor whereas albumin administration was not effective. Possible structure and action of the noncovalently bound albumin and bilirubin complex are discussed.

[The effect of chronic butyphos poisoning on protein and nucleic acid synthesis in various rat organs and activity of blood cholinesterase]. / Vliianie khronicheskoi intoksikatsii butifosom na sintez belka i nukleinovykh kislot v razlichnykh organakh krys i aktivnost' kholinésterazy krovi.

Two doses of butyphos (1/10 and 1/50 LD50) were administered into rats within 6 months. Content of DNA was distinctly decreased in liver and kidney tissues within the first 2 weeks of the pesticide administration. Then DNA synthesis was increased 2-fold in liver tissue and remained high during all the 6 months of intoxication. Protein synthesis was increased in liver tissue within 3 months of the administration and remained elevated up to the end of experiment. High rate of protein synthesis, found in kidney and spleen tissues at the initial steps, was markedly decreased within 6 months. Content of DNA and RNA was decreased in the tissues studied within 1 month of the intoxication and restored within 3 months, while it remained at considerably lower level in liver and spleen tissues as compared with control values. Cholinesterase activity was lowered by 90% in blood within 11 weeks with the subsequent increase; but in the experimental group intoxicated with butyphos at 1/10 LD50 the enzymatic activity constituted only 60% of control values within 6 months. Histological study showed development of necrodystrophy in liver tissue and of fibroplastic glomerulonephritis in kidney. The deteriorating effect of butyphos on cellular genome functions appears to relate not only to its cytotoxicity but also to the cancerogenic and mutagenic properties of the pesticide.

Mitogenic effect of kallikrein from human urine on cultured human skin fibroblasts. Analysis of the combined action of kallikrein, insulin, and fibroblast growth factor on DNA and RNA synthesis.

Kallikrein isolated from human urine was capable of stimulating DNA and RNA synthesis in cultured human skin fibroblasts in media with a low serum content. The same concentration of kallikrein had a different effect on the DNA and RNA synthesis in different fibroblast lines, which was attributed to differences in the sensitivity of the cells to kallikrein. At a dose of 0.5 micrograms ml-1, kallikrein inactivated by heating at 100 degrees C caused an abrupt decrease in DNA synthesis in all the cell lines studied. When either active or inactivated kallikrein was added to the growth medium simultaneously with insulin there was a competitive effect on DNA and RNA synthesis. Preincubation of the cells with kallikrein prior to addition of insulin led to a reduction in the level of DNA synthesis compared to that seen upon simultaneous addition of kallikrein and insulin, suggesting that kallikrein and insulin competed for the same receptor. When kallikrein and fibroblast growth factor (FGF) were added simultaneously to the growth medium, there was a sharp decrease in both DNA and RNA synthesis in the cells compared to that seen on addition of FGF alone. Since heparin protected FGF from kallikrein inactivation, it is suggested that inactivation was caused by proteolytic degradation of part of the FGF molecule by kallikrein. It is concluded that kallikrein and insulin compete for the same receptor, possibly the insulin-like growth factor I (IGF-I), and that binding of kallikrein to this receptor is a prerequisite for mediation of the stimulatory effect of kallikrein on nucleic acid synthesis.

[The regulation of DNA synthesis by fibronectin and its proteolytic products in the skin fibroblasts of healthy donors and patients with systemic scleroderma and rheumatoid arthritis]. / Reguliatsiia sinteza DNK fibronektinom i produktami ego proteoliza v fibroblastakh kozhi zdorovykh donorov i bol'nykh sistemnoi sklerodermiei i revmatoidnym artritom.

To study the effect of fibronectin isolated from plasma and culture media and the effect of its tryptic hydrolyzates on DNA synthesis, cultured skin fibroblasts of healthy donors and these of patients with systemic scleroderma (SSD) and rheumatoid arthritis (RA) were employed. It was shown that both fibronectin and total products of its proteolysis markedly stimulated DNA synthesis only in skin fibroblasts of patients with SSD. Fibronectin fragments inhibited DNA synthesis in all fibroblast strains studied. The effect of fibronectin and all its Gel fragments on the DNA synthesis in skin fibroblasts of patients with SSD was dose-dependent. The activity of total fibronectin tryptate, Gel-fragment-free tryptate, and Gel fragments themselves depended on the duration of fibronectin proteolysis, i. e. on the size of the fragments obtained. Culture media collected after treatment of fibroblast monolayer with trypsin and subsequent removal of fibronectin Gel fragments had mitogenic effect on skin fibroblasts, especially on those of patients with SSD and RA. It is supposed that fibronectin Gel fragments are inhibitors of growth factors produced by fibroblasts. The results suggest that fibronectin and its fragments have an important regulatory role in fibroblast proliferation.

[Stimulation of DNA and RNA synthesis in the liver of hepatectomized rats by fractions of liver cytosol and conditioned media obtained during cultivation of rat liver explants]. / Stimuliatsiia sinteza DNK i RNK v pecheni gepatéktomirovannykh krys fraktsiiami tsitozolia pecheni i konditsionirovannoi sredy, poluchennoi pri kul'tivirovanii éksplantatov pecheni krys.

Heat resistant fractions stimulating proliferation (PSF) were identified in the fractions of rat liver cytosol after 70% hepatectomy as well as in the medium harvested after cultivation of rat liver explants. The PSF augmented considerably the rate of DNA synthesis, it increased synthesis and transport of rapidly labelled RNA as well as the level of mitotic activity in rat liver tissue resected by 30% and 80%. Role of the PSF in regulation and acceleration of regeneration processes in the residual parts of liver tissue, remained after hepatectomy, is discussed.

[Metabolism of glycoproteins and fibronectin in skin fibroblasts of patients with rheumatoid arthritis]. / Metabolizm glikoproteinov i fibronektina v fibroblastakh kozhi bol'nykh revmatoidnym artritom.

The distribution in the cellular monolayer of the de novo synthetized pre-labeled glycoproteins and fibronectin upon culturing of fibroblasts in the medium with low serum content was analyzed. It was found that in rheumatoid arthritis (RA) the amount of total glycoproteins on the surface and within fibroblasts is higher and in the extracellular matrix is lower than in skin fibroblasts of healthy donors (HD). However, the amount of pre-labeled fibronectin on the surface of skin fibroblasts from patients with RA was considerably lower than in those from HD This finding as well as a rapid decrease in the amount of pre-labeled fibronectin in the extracellular matrix of RA fibroblasts is indicative of a more rapid metabolism of this protein in RA. In the skin fibroblasts from HD there was a practically uniform decrease in the amount of pre-labeled fibronectin in the cellular monolayer. The presence of caseinolytic activity in the culture medium even upon the first day of cell culturing in the serum-free medium, as well as the effect of various proteinase inhibitors on glycoprotein content in the cellular monolayer provide evidence that the rate of glycoprotein and fibronectin metabolism, especially in connective tissue cells of patients with RA, might possibly be determined not only by the level of their synthesis but also by the level of proteolytic activity in the connective tissue cells.

[Effect of a medium with a low serum content on protein synthesis and secretion and RNA and DNA synthesis in a skin fibroblast culture from patients with rheumatoid arthritis and systemic scleroderma]. / Vliianie sredy s nizkim soderzhaniem syvorotki na sintez i sekretsiiu belka, sintez RNK i DNK v kul'ture fibroblastov kozhi bol'nykh revmatoidnym artritom i sistemnoi sklerodermiei.

Synthesis and secretion of protein, as well as synthesis of RNA and DNA by skin fibroblasts of patients with systemic sclerodermia (SSD) and rheumatoid arthritis (RA) upon prolonged culturing of fibroblasts in the medium with low (0.5-1%) serum content differ markedly in their direction and intensity from protein, RNA and DNA synthesis by skin fibroblasts of healthy donors (HD) and by fetal fibroblasts. It has been found that skin fibroblasts of patients with RA and SSD, as well as those of HD, secrete 75-80% of protein synthesized by fibroblasts de novo upon their culturing in DMEM medium with 1% human serum. Under the same conditions, on days 2-5 of culturing, RNA synthesis in the fibroblasts of patients with RA and SSD was increased 3-4-fold, while DNA synthesis was increased 2-3-fold. Collagenolytic and caseinolytic activity in the culture medium of skin fibroblasts from HD and patients with RA reached maximal levels on days 3-5. High protein secretion was observed in DMEM serum-free medium in the presence of vitamin mixture upon culturing skin fibroblasts of patients with SSD. The results obtained show that skin fibroblasts from HD differ in their functional activity from those of patients with rheumatic disorders. It might be suggested, therefore, that the mechanism of protein secretion plays an important role in the maintenance of constant intracellular protein levels in resting cells.

[Protein synthesis and secretion and RNA and DNA synthesis in human skin fibroblasts in a medium with a low serum content]. / Sintez i sekretsiia belka, sintez RNK i DNK u fibroblastov kozhi cheloveka v srede s nizkim soderzhaniem syvorotki.

Human skin fibroblasts, both postnatal and embryonic, were cultured in the stationary phase of growth for 6-10 days in the DMEM with bovine serum (BS), 0.1-0.5% fetal calf serum (FCS) or 1% human serum (HS). On the day 4 of culturing, a considerable increase was observed in the synthesis and secretion of protein by postnatal fibroblasts in the Eagle medium with 0.1-0.5% FCS, or with 0.5% BS, and in medium 199 with 0.1-0.5 BS, or with 0.1 FCS. Maximum synthesis and secretion of 14C-proline labeled protein was observed on day 2 of culturing of cells in the DMEM medium with 1% HS. In the DMEM medium with low serum content, protein synthesis being virtually unchanged, 75-80% of protein was secreted by cells into the culture medium with BS on days 2-4; in the medium with FCS such a high secretion of protein was observed only on day 4. High synthesis of protein by fetal fibroblasts in the DMEM medium with 0.1% BS and high protein secretion in all the media with 0.1% BS or 0.5% FCS were observed. The maximum level of secretion of protein by fibroblasts coincided with a considerable increase in both RNA and DNA syntheses. The data obtained suggest that cells in deep resting state actively react to the composition of the medium as well as to the quality and quantity of the serum. It may also be suggested that the mechanism of protein secretion has an important role in maintenance of the constant level of intracellular proteins in resting cells.

[Protein, RNA and DNA synthesis in skin fibroblast cultures from healthy donors and patients with rheumatic diseases]. / Sintez belka, RNK i DNK v kul'ture fibroblastov kozhi zdorovykh donorov i bol'nykh revmaticheskimi zabolevaniiami.

Synthesis of protein, RNA and DNA was studied in skin fibroblast cultures of healthy donors and patients with systemic scleroderma (SSD) and in those with rheumatoid arthritis (RA) with the use of 14C-protein hydrolyzate, 14C-uridine and 14C-thymidine, respectively. A study was also made of the stimulation of 14C-proline incorporation in protein fibroblasts upon addition to serum-free media of 5% bovine embryonic serum. The stability of RNA in fibroblasts was tested. It was shown that the rate of protein synthesis was 11 times higher in fibroblasts of RA patients and 6 times higher in those of SSD patients as compared to the rate of protein synthesis in fibroblasts of normal subjects. The rate of DNA synthesis in skin fibroblasts of RA patients was 15 times higher and in those of SSD patients 4 times higher than normal. In both RA and SSD patients, the synthesis of short-labeled RNA was 2-3 times higher than normal. The addition of embryonic serum increased 2-3 times the incorporation of 14C-proline in protein skin fibroblasts of SSD patients. It was found that all RNA in skin fibroblasts was represented by long-living molecules and that 30-40% of short-labeled RNA in skin fibroblasts of healthy donors and SSD patients underwent degradation within 1-2 hours. The data obtained indicate that fibroblasts of the two pathologies under study are characterized by considerable differences in the synthesis of DNA and the activity of the protein-synthesizing system.

[Synthesis of protein, RNA and DNA and alkylation of macromolecules in the cells of sensitive and resistant strains of ascites hepatoma 22a induced by N-methyl-N-nitrosourea]. / Sintez belka, RNK, DNK i alkilirovanie makromolekul v kletkakh chuvstvitel'nogo i rezistentnogo shtammov astsitnoi gepatomy 22a pri deistvii N-metel-N-nitrozomocheviny.

It was shown that both in vivo and in cell suspension cultures of N-methyl-N-nitrosourea (MNM)-sensitive and resistant strains of hepatoma 22a the alkylation of macromolecules by [14C]MNM and radioactivity in the acid-soluble cell fraction and ascite fluid at different time intervals after the label injection are practically the same. The synthesis of protein, RNA and DNA in the resistant strain cells is either not impaired at all or is rapidly restored after MNM injection in vivo. The mechanism of resistance is discussed in terms of cell ability to restore DNA damage and of the lack of cell sensitivity to target cell damage due to alkylation.

[Metabolic activation of N-methyl-N-nitrosourea in tumor cells]. / Metabolicheskaia aktivatsiia N-metil-N-nitrozomocheviny v opukholevykh kletkakh.

An enzymatic activation of N-methyl-N-nitrose urea (MNU) was studied in microsomes, isolated from hepatoma 22a ascites cells, using a procedure which enabled to reveal the MNU enzymatic activation in mice liver microsomes. The MNU metabolic activation was not observed in hepatoma cell microsomes; at the same time, the MNU activation occurred when the cells were incubated under conditions of a suspension culture. The enzymes, participating in metabolic activation of MNU, were apparently arranged at the surface of the tumoral cells. Alkylation of MNU macromolecules was shown to be a two-step process. At the first step alkylation was due to spontaneous degradation of MNU and at the second step it was responsible for formation of active products as a result of MNU metabolic activation. MNU appears to induce synthesis of enzymes required of its metabolic activation in hepatoma 22a cells within 30 min.

[Accelerated microsomal DNA synthesis under the influence of xenobiotics and chemical carcinogens]. / Uskorenny sintez mikrosomnoi DNK pod vliianiem ksenobiotikov i khimicheskikh kantserogenov.

Injection of 3,4-benz(a)pyrene, methyl nitrosourea and phenobarbital into healthy mice of the C3HA line results in a rapid, sharp increase of [14C]-thymidine incorporated into liver microsomal DNA, accompanied by a suppression of nuclear DNA synthesis. In the liver of neoplastic mice and in the ascite cells of hepatoma 22A the system of microsomal DNA synthesis was insensitive to the injection of methyl nitrosourea. Cycloheximide and puromycin, which strongly inhibited nuclear DNA synthesis, had no effect on the synthesis of microsomal DNA. Stimulation of [14C]-thymidine incorporation into microsomal DNA after injection of methyl nitrosourea and 3,4-benz(a)pyrene may be accounted for not only by an increase of the DNA reparation processes, since caffeine, the inhibitor of post-replicatory reparation of DNA, did not eliminate the induction of microsomal DNA synthesis in the liver. Hydroxyurea in combination with methyl nitrosourea and phenobarbital significantly suppressed the synthesis of nuclear DNA in the liver and did not affect the synthesis of mtDNA; the stimulating effects of these inducers on the synthesis of microsomal DNA was thereby removed. This is indicative of independence of synthesis of microsomal DNA on that of nuclear DNA and mtDNA. Different specific radioactivities of microsomal, nuclear and mtDNAs in the regenerating mouse liver on the 5th, 10th and 15th post-hepatectomy days may be due to different metabolic stability of these DNAs. A possible role of microsomal DNA as a xenobiotic system component is discussed.