Influence of exogeneous proteins on mutagenic process

Mutagenic factors of biological origin by the example of carbohydrate-binding proteins are observed. An attempt of summarizing the existing data concerning participation of exogenous macromolecules in mutagenic process is made. The mechanisms of the influence of exogenous macromolecules on mutagenesis, proliferation, and surviving of mammalian cells are discussed.     

Stimulation of cartilage macromolecule synthesis by adenosine 3',5'-monophosphate.

The role of cyclic AMP in the regulation of cartilage macromolecule synthesis in vitro was studied in pelvic cartilage from 10-12 day chick embryos. Incubation of cartilages in medium containing 0.5 mM cyclic AMP resulted in a 30% inhibition of 35SO4-2, [3H]leucine and [3H]uridine incorporation into proteoglycan, total protein and RNA, respectively. Higher concentrations of cyclic AMP had no greater effects. In contrast, butyrylated cyclic AMP derivatives (0.5-5.0 mM) added to the incubation medium stimulated (50-100%) the incorporation of these radiolabeled precursors into cartilage macromolecules. Theophylline, in concentrations (0.1-0.5 mM) which raise intracellular cyclic AMP, also increases the incorporation of radiolabeled precursors into macromolecules. The data indicate that exogenous cyclic AMP and butyrylated cyclic AMP derivatives have paradoxical effects on cartilage macromolecule synthesis. Butyrylated cyclic AMP derivatives, not exogenous cyclic AMP, mimic the effects of intracellular cyclic AMP. Incubation of embryonic chicken cartilage with exogenous cyclic AMP results in the extracellular degradation of the cyclic AMP to adenosine. Adenosine (0.125 mM) inhibits precursor incorporation into cartilage macromolecules. The metabolism of exogenous cyclic AMP generates sufficient adenosine to account for the observed inhibitory effects of exogenous cyclic AMP on cartilage macromolecule synthesis. Butyrylated cyclic AMP derivatives are not degraded during incubation with cartilage. The data indicate that cartilage is a tissue in which the effect of cyclic AMP is to stimulate anabolic processes.     

Crystallographic analysis of a full-length streptavidin with its C-terminal polypeptide bound in the biotin binding site

The structure of a full-length streptavidin has been determined at 1.7 A resolution and shows that the 20 residue extension at the C terminus forms a well-ordered polypeptide loop on the surface of the tetramer. Residues 150-153 of the extension are bound to the ligand-binding site, possibly competing with exogenous ligands. The binding mode of these residues is compared with that of biotin and peptidic ligands. The observed structure helps to rationalize the observations that full-length mature streptavidin binds biotinylated macromolecules with reduced affinity.     

Sulphate turnover of surface proteoglycans in cultured rat smooth muscle cells

Turnover of radioactive sulphate-labelled proteoglycans in cultured rat smooth muscle cells was detected by pulse chase techniques. The degradation appeared to take the form of desulphation of sulphated macromolecules, with a loss in total sulphate of approximately 50% in 5 days. The desulphation process occurred in the pericellular/matrix compartment of the culture system and was unaffected by inhibition of matrix formation by beta-aminopropionitrile, or by incubation of cells with lysomotropic inhibitors. There was no evidence for further degradation of desulphated species even when exogenous, radio-labelled proteoglycans were added to fresh cultures and incubated for four days. Labelled macromolecules initiated on xyloside acceptors were desulphated by rat smooth muscle cell cultures more slowly than intact proteoglycans.     

Protein microinjection by protease permeabilization of fibroblasts

Exposure of cultured diploid fibroblasts to protease solutions induces a hyperpermeable state which permits entry of exogenous macromolecules directly into the cytosol. We have exploited this finding to devise a microinjection method whose chief advantages are simplicity and good retention of cell viability. Proteins successfully injected by this technique range from insulin to thyroglobulin. The amounts injected range from 4 X 10(5) to 5 X 10(6) molecules/cell.     

Minicells of Bacillus subtilis a unique system for transport studies

Ultrasound-purified minicells produced by Bacillus subtilis mutant div IV-Bl have been studied for their ability to transport and incorporate into macromolecules a variety of amino acids, uracil and thymine. Minicells transport all 12 amino acids examined, but are unable to incorporate them into macromolecules. No significant differences were found in the initial uptake rates of glutamic acid, aspartic acid, and alanine by minicells and parental cells. The uptake of methionine and proline by minicells was shown to be inhibited by metabolic poisons, indicating an energy-metabolism requirement for transport in this system. The proline pool in minicells was found to be readily exchangeable with exogenous proline. In contrast metabolically poisoned minicells only slowly lose their pool proline, indicating an energy requirement for pool maintenance. Packed-cell experiments reveal that minicells accumulate proline against a concentration gradient.In addition to amino acids, minicells are able to transport uracil but cannot incorporate uracil into acid-precipitable material (RNA). Neither thymine transport nor its incorporation into macromolecules can be demonstrated in minicells.Minicells appear to be a new system, therefore, in which transport may be studied in the absence of macromolecular biosynthesis.     

Ultraviolet light induced alteration to the skin

Solar light is the primary source of UV radiation for all living systems. UV photons can mediate damage through two different mechanisms, either by direct absorption of UV via cellular chromophores, resulting in excited states formation and subsequent chemical reaction, or by phosensitization mechanisms, where the UV light is absorbed by endogenous (or exogenous) sensitizers that are excited and their further reactions lead to formation of reactive oxygen species (ROS). These highly reactive species can interact with cellular macromolecules such as DNA, proteins, fatty acids and saccharides causing oxidative damage. Direct and indirect injuries result in a number of harmful effects such as disrupted cell metabolism, morphological and ultrastructural changes, attack on the regulation pathways and, alterations in the differentiation, proliferation and apoptosis of skin cells. Processes like these can lead to erythema, sunburn, inflammation, immunosuppression, photoaging, gene mutation, and development of cutaneous malignancies. The endogenous and exogenous mechanisms of skin photoprotection are discussed.     

The Effect of Histones and Other Basic Macromolecules on Cell Permeability and Elongation of Barley Roots

Low exogenous concentrations of calf-thymus histone, poly-l -lysine and lysozyme inhibit root elongation. These basic macromolecules (polycations) also affect cell permeability resulting in a leakage of ultraviolet light (UV)-absorbing materials and chloride ion from cells. Adenosine 5′-monophosphate was identified as one of the UV-absorbing compounds in the root exudate, by thin layer chromatography. These deleterious effects of polycations on root growth and permeability are reduced in the presence of calcium and other divalent cations. Calcium ion-histone interaction appears to exhibit competitive kinetics and suggests that both calcium and histone compete for attachment to negative sites on cell membrances.     

Stimulation of replicative DNA synthesis by eukaryotic proteins bound to single-stranded DNA]

The paper deals with the effect of the single-strand (ss) DNA-binding proteins (SSB-proteins) from the Ehrlich ascites tumor (EAT) cells and from the eggs of silkworm, as well as the mouse serum blood proteins, having preferential affinity to ss DNA, on the DNA replicative synthesis in the EAT cells permeable for the macromolecules, and, for the silkworm proteins and on the DNA replicative synthesis in the nuclei from the eggs of silkworm proteins and on the DNA replicative synthesis in the nuclei from the eggs of silkworm permeable for macromolecules. SSB-proteins of EAT to considerable extent stimulated the DNA synthesis. At the same time, the other proteins (from the silkworm and from the serum) activated the DNA synthesis in the permeable cells to the less extent. It was found that SSB-proteins from the silkworm had a 1.5-13 fold stimulating effect on the DNA replicative synthesis in the homologous system (in the permeable nuclei). If the permeability for the macromolecules of the cells and nuclei treatment with Triton X-100 may be different, it is supposed that the activation of the DNA synthesis by the exogenous proteins depends on the homologous system of the DNA replicative complex. It is possible that the effect of the serum proteins on the DNA synthesis is connected with the masking of the ss regions of DNA which inhibited DNA-polymerase alpha. Perhaps the mechanisms of the activation of the DNA replicative synthesis by the proteins in vitro with the purified DNA polymerase alpha and in vivo are of different nature and are conditioned by homology of the deoxyribonucleoproteins.     

An edeine resistant mRNA-dependent protein synthesis system from a Saccharomyces cerevisiae mutant

A cell-free protein synthesizing system from a mutant of Saccharomyces cerevisiae translated exogenous mRNA in the presence of 2 microM edeine, while a similar system from wild-type strain was completely inhibited by the drug. The mutant ribosomes showed an affinity for [125I]edeine comparable to the wild-type ribosomes, thereby suggesting that these macromolecules alone were not responsible for the edeine-resistant capacity of the mutant.     

The uptake of endogenous proteins by oocyte nuclei

Late stage 5, and stage 6 Xenopus oocytes were micro-injected with tritiated leucine or proline. Incorporation of the isotope into TCA-precipitable material was complete in approx. . At intervals of , 1, 3, and 6 h after injection, nuclei were isolated by hand and fixed in ethanol. The nuclear envelopes were removed, and the size distribution of labelled polypeptides within the remaining nucleoplasm was determined on SDS-polyacrylamide gels. Labelled polypeptides ranging in molecular weight from about 11000 to 150000 were present in the nucleus h after injection. With increasing time, the number of counts per nucleus increased, as did the proportion of larger polypeptides. Using puromycin, it could be demonstrated that the incorporated macromolecules were taken up from the cytoplasm, and not synthesized in the nucleus. After 3 h the concentration of labelled polypeptides having molecular weights of 94 000 and above was about four times greater in the nucleus than in the cytoplasm. These rates of nuclear accumulation of macromolecules are greater than would have been predicted from previous studies of nuclear permeability utilizing exogenous tracers. Possible explanations for these differences are considered.     

Polyamines in murine splenic lymphocytes

Diacetyldiamines cause compromised B-lymphocyte function, as evidenced by our previous demonstration of inhibition of mitogen activation and decreased secretion of immunoglobulin in murine spleen cultures. In this study, we report that putrescine and spermidine are differentially metabolized by the cell. Diacetyldiamine, which is also taken up by these cells and metabolized, causes a striking decrease in cell uptake of exogenous putrescine and spermidine. We also report for the first time that several distinct macromolecules containing radioactive polyamines may be resolved, and that hypusine is present in more than one species of macromolecule.     

Comparison of fixatives for maximal retention of radiolabeled glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated [35S]-sulfate

Previous studies have used [35S]-sulfate as a specific marker to autoradiographically localize sulfated glycosaminoglycans, proteoglycans, and glycoproteins. Embryonic chicks were labeled with [35S]-sulfate, followed by previously reported routine fixation and processing techniques. Subsequent processing revealed loss of radiolabeled macromolecules and retention of unincorporated label in the tissue, using these procedures. Biochemical analysis after various fixation and processing procedures demonstrated that an additional agent, such as cetylpyridinium chloride, was necessary in the fixative to retain the highly aqueous soluble sulfated macromolecular components. Molecular sieve chromatography was used to monitor digestate solutions for the identity of glycosaminoglycans and proteoglycans as indicated by selective enzymatic removal. Retained unincorporated [35S]-sulfate could be completely removed by rinsing the tissue in dehydration solutions containing exogenous sodium sulfate. This new procedure ensures the quantitative retention of sulfate labeled macromolecules in fixed tissue with the complete removal of unincorporated radiotracer, both of which are necessary for meaningful autoradiography.     

蛋白质转导及其内在化机制

蛋白质转导是新近发展起来的向细胞内快速输送外源性大分子或高极性分子的有效途径。它实质上是一些蛋白质,尤其是病毒蛋白上被称为蛋白质转导区(PTD)的小片段,蛋白质和其他物质,如DNA、脂质体、纳米颗粒、环孢素A等与之结合后,即能够被携带进入细胞或穿过血脑屏障。蛋白质转导的内在化机制目前尚不清楚,可能与带正电荷(富Arg)的PTD肽与细胞膜上带负电荷的硫酸乙酰肝素有关,但不排除其他内在化机制。     

Cell wall permeability and export of proteins into the culture broth of Saccharomyces cerevisiae

The upper limit of Saccharomyces cerevisiae cell wall permeability is such that only molecules with a mass not greater than 2-3 kDa can pass across the cell wall. However, proteins with a much higher molecular mass are exported into the cultural broth when the organism is cultivated in a liquid medium with peptone. Under certain conditions, exogenous proteins interact with the cytoplasmic membrane. These as well as some other findings imply that the cell wall may have microplots with an anomalous permeability, via which the regulated exchange of macromolecules is realized between the intracellular and outer media.     

Foreign protein can be carried into the nucleus of mammalian cell by conjugation with nucleoplasmin

In studies on the specific migration of macromolecules across the nuclear envelope, a karyophilic protein was injected into the cytoplasm of cultured cells and its subsequent location in the cell was examined. Nucleoplasmin of frog nuclear protein was used for this experiment. When [125I]nucleoplasmin was introduced into the cytoplasm of mammalian cells (human and mouse) by red blood cell-mediated microinjection, it rapidly accumulated in the nucleus. When nucleoplasmin conjugated with [125I]IgG against chromosomal protein was introduced similarly, it also accumulated rapidly in the nucleus, and reacted with its antigen inside the nucleus. On the contrary, when IgG alone or IgG conjugated with BSA were introduced, they did not migrate from the cytoplasm into the nucleus. These findings imply that the migration of macromolecules from the cytoplasm to the nucleus does not depend only on their molecular size but also on a specific transport mechanism, and that karyophilic proteins may act as useful carriers in the transfer of exogenous proteins into the nucleus.     

Endocytic pathways and time sequence of lysosomal transfer of macromolecules in cultured mouse peritoneal macrophages

Summary A double-labeling protocol was used to study endocytic pathways and lysosomal transfer of exogenous macromolecules in cultured mouse peritoneal macrophages. After pulse-chase labeling of lysosomes with horseradish peroxidase (visualized cytochemically), the cells were exposed to native, anionic ferritin for 0–45 min at 37° C and then analysed by transmission electron microscopy. The results show that ferritin binds to the plasma membrane, accumulates in coated pits, and is rapidly taken up in small, smooth-surfaced endocytic vesicles. The latter carry the ferritin molecules directly to lysosomes, recognized by their peroxidase labeling, or fuse with each other to form larger endocytic vacuoles (endosomes) which in turn fuse with and empty their content into lysosomes. The first signs of transfer of ferritin into the lysosomes were seen after 5–10 min of exposure and after 25–30 min most of the lysosomes were labeled. Union of ferritin-labeled and other lysosomes was also noted, suggesting that the contents of the lysosomes were spread within the lysosomal compartment by fusion-fission processes. It is concluded that a multiplicity of structures is involved in the uptake and intracellular transport of exogenous macromolecules in macrophages and that the time sequence of lysosomal transfer of the interiorized material is highly variable.     

A method for incorporating macromolecules into adherent cells

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.     

Glycan profiling of gel forming mucus layer from the scleractinian symbiotic coral Oculina arbuscula

The gel forming mucus layer surrounding scleractinian corals play fundamental functions in the maintenance of a favorable microenvironment required for the survival of these organisms. In particular, it harbors a rich partially species-specific symbiotic community through yet poorly understood molecular interactions. However, removal or contamination of this community by exogenous bacteria is closely linked to the worldwide bleaching events that are presently devastating coral colonies. The present study investigates the structure of major high molecular weight glycoconjugates that are responsible for both rheological properties of mucus and sugar-protein interactions with microbial communities. We demonstrated that it is composed by two distinct types of sulfated macromolecules: mucin type glycoproteins densely substituted by short unusual O-linked glycans and repetitive polysaccharides.