A consideration of the role of cell surface macromolecules in the process of viral transformation.

There is extensive physiological evidence implicating the cell surface as the key organelle which mediates the cell:cell interactions which underlie both normal and neoplastic growth. This information has now been supplemented with biochemical and biophysical data which indicates that surface macromolecules, in particular the heteroglycans of transformed cells, differ from those which lie at the periphery of normal cells. In the case of cells neoplastically transformed by most tumour viruses it is clear that the small virus genome (2-5 x 10(6) daltons) cannot carry the total genetic information to accomodate these various biochemical modifications, if indeed they are encoded in separate genes (1). To examine the part played in transformation by cellular genes coding for surface heteroglycan formation, we have turned to a study of SV-3T3 cells (ts H6-15) which are temperature-sensitive for expression of the transformed cell phenotype (2). The data show that cells grown under conditions permissive and non-permissive for such expression exhibit the same pattern of formation of glycolipids, and the majority of the polypeptides of the plasma membrane. There are, however, significant differences in the synthesis of some glycopeptides. A large molecular weight, trypsin-labile glycopeptide, present at the surface of untransformed fibroblasts but barely measurable in some of their virus-transformed derivatives (3), was detected, essentially at the same level, at the surface of ts H6-15 cells grown at the permissive and non-permissive temperatures. The signficance of these observations is discussed.     

Mapping the surface properties of macromolecules.

Methods are presented for the rapid computation of schematic projections of the surfaces of macromolecules, similar to the "roadmaps" used to illustrate the surfaces of viruses (Rossmann, M.G. & Palmenberg, A.C., 1988, Virology 164, 373-382). Several types of projections are described, extending the application of "roadmaps" to the external surfaces of all macromolecules and their interior binding pockets and pores. The surface projections, showing the positions of residues, can be colored, shaded, contoured, and annotated to show physical, sequence, or functional properties such as surface topology, hydrophobicity, or sequence conservation, for example. The automated procedures are useful for surveys of the surface features of proteins sharing similar functional properties.     

Permeability of cyanobacterial mucous surface structures for macromolecules

The space of diffusive distribution of neutral hydrophilic macromolecules (dextrans with molecular sizes of 1.5 to 9 nm in the Stokes’ radius values) in the mucous surface structures (MSS) of intact bacterial cells has been studied for the first time on cyanobacteria. Cyanobacterial species and strains under study belong to different taxonomic groups, the members of which form MSS of various morphology and ultrastructure and can grow in association with plants and animals, inter alia as mucous microcolonies. The range of permeability has been determined by the fractionation of polydisperse dextrans method, previously applied for plants, in combination with electron microscopy. Dextrans are supposedly distributed in the MSS polysaccharide matrix in accordance with their sizes, in much the same way as in a macroporous unitary gel. The similarity of the chemical composition and macromolecular organization of cyanobacterial MSS with pectins of plant cell walls and the role of MSS and the intercellular matrix as permeability barriers in associative interactions of microorganisms are under consideration.     

Possible role of genetic cell variants in the viral induction of mouse mammary tumors

Summary Out of three attempts to induce neoplasia in normal C57B1 mammary epithelial cells with the mouse mammary tumor virus (MuMTV) only one presented signs of tumorigenicity. Immunofluorescence showed that virus synthesis took place in all three sublines but tumorigenicity as detected by cell aggregation viability (CAV) and transplantation into syngeneic mice failed to occur in two of them. By comparison, cells from a BALB/c spontaneous mammary tumor that do not express MuMTV were 100% tumorigenic, whereas cells from a BALB/cfC3H tumor with a 95% virus-producing cell population had a normal CAV and were tumorigenic only in 60% of the test animals. This lack of correlation suggested that many of the virus-producing cells were not neoplastic and that neoplasia might occur under virus stimulation only if a restricted population of genetic cell variants existed. Accelerated tissue culture passages of virus-free C57B1 and BALB/c normal mammary cells resulted in their spontaneous neoplasia at Passages 23 and 50 respectively; when duplicated cells cryopreserved in early passages were revived and cultivated in the same manner, neoplasia occurred at Passages 27 and 58. The similarity of the passage numbers appears to confirm the existence of genetic cell variants among the normal cell population. This investigation was supported by U.S. Public Health Service Grant R01-CA-08515 from the National Cancer Institute.     

Imitational modeling of process of evolution: From organic macromolecules to protocell and animal cell

A dynamic imitational model of initial stages of cell evolution has been developed based on role of environmental calcium concentration. The model is designed from our hypothesis about the medium of the appearance of protocells, which could be potassium water reservoirs rather than sea salt water with its predominance of sodium salts. The necessary elements of the appearance of the protocells served organic molecules, code of their synthesis, and formation of macromolecules under favorable ion concentration in environment: a high K⁺ and Mg²⁺ and a low Na⁺ concentration. The model is based on an assumption that one of the first stages in evolution of life was the appearance in the potassium-magnesium water reservoirs of organic molecules capable for self-replication on the basis of genetic code and formation of protocell with the potassium cytoplasm. The model has demonstrated necessity of formation of cell envelope for development of the protocell. Replacement of the dominant cation in water reservoirs—potassium by sodium—required the appearance of ion-transporting devices in plasma membrane and their participation in adaptation of cells to environment. This stage of evolution was accompanied by the most important morphofunctional event—formation of the plasma membrane instead of cell envelope. The membrane provided the ion asymmetry in the cell (preservation of K⁺ in it) relatively to the sodium external medium for maintaining optimal intracellular medium. In the model system, predecessors of animal cells elaborated mechanism of maintenance of the potassium cytoplasm with the sodium counterion dominating in the environment.     

A method for concentrating dilute solutions of macromolecules

A simple, inexpensive acrylate polymer which has a capacity to absorb 170 ml of water per g has been developed. It can be used to concentrate dilute solutions of macromolecules such as proteins, nucleic acids, and carbohydrates. The polymer absorbs only low-molecular-weight substances such as glucose, sucrose, and inorganic salts. It can replace the various conventional concentration methods. No special device or electricity is needed for the concentration. The inexpensive polymer, molded in the form of rods, can be very conveniently used as “disposable concentration sticks.”     

Increased trypsin sensitivity of cell surface macromolecules after malignant transformation.

The effect of treatment with 0.04% (w/v) trypsin (EC 3.4.4.4) for 3 h on the electrophoretic mobility (EPM) of polyoma-virus malignantly transformed BHK21 cells (Py6) and their normal counterparts has been investigated. These particular conditions were chosen because an earlier study had shown that such treatment released material from the Py6 cells which was not obtained from the BHK21 cells. The negative EPM of the Py6 cells at pH 7.5 was greatly increased by this treatment; whereas the EPM of the BHK21 cells remained unchanged. Active enzyme was required to produce the change. No evidence was obtained for cytolysis, cytotoxicity or uptake of the enzyme by the treated Py6 cells. Measurement of the EPM of the Py6 cells at different pH levels before and after trypsin treatment suggested that the enzyme was removing cationic groupings from the cell surface.     

Nanoscale amphiphilic macromolecules as lipoprotein inhibitors: the role of charge and architecture

A series of novel amphiphilic macromolecules composed of alkyl chains as the hydrophobic block and poly(ethylene glycol) as the hydrophilic block were designed to inhibit highly oxidized low density lipoprotein (hoxLDL) uptake by synthesizing macromolecules with negatively charged moieties (ie, carboxylic acids) located in the two different blocks. The macromolecules have molecular weights around 5,500 g/mol, form micelles in aqueous solution with an average size of 20-35 nm, and display critical micelle concentration values as low as 10(-7) M. Their charge densities and hydrodynamic size in physiological buffer solutions correlated with the hydrophobic/ hydrophilic block location and quantity of the carboxylate groups. Generally, carboxylate groups located in the hydrophobic block destabilize micelle formation more than carboxylate groups in the hydrophilic block. Although all amphiphilic macromolecules inhibited unregulated uptake of hoxLDL by macrophages, inhibition efficiency was influenced by the quantity and location of the negatively charged-carboxylate on the macromolecules. Notably, negative charge is not the sole factor in reducing hoxLDL uptake. The combination of smaller size, micellar stability and charge density is critical for inhibiting hoxLDL uptake by macrophages.     

The role of transformation in the variability of the Neisseria gonorrhoeae cell surface

This review discusses the genetic basis for surface changes in Neisseria gonorrhoeae and the role of specific transformation reactions in producing them. Variation in the structure of pilin, the subunit of gonococcal pili, occurs by transformation-mediated recombination of DNA segments in storage loci with the expression locus. These pilin loci have low recombination potential since their sequences contain only short uninterrupted identical sequences. The DNA within storage or silent loci are also relatively deficient in the short defined sequences which target DNA for efficient uptake and thus have relatively low affinity for the DNA transport system. Consequently, pilin-encoding DNA segments constitute relatively poor substrates for the general transformation system of gonococci. These considerations suggest the existence of locus-specific factors which increase the efficiency of genetic exchange between pilin loci. I raise the speculative hypothesis that one function of transformation-mediated DNA entry is to provide a regulatory stimulus signalling the death of neighbouring gonococci. This regulatory shift might lead to production of factors which accelerate genetic reshuffling of pilin loci either by transformation per se using external DNA as donor, or via a recombinational process which utilizes internally derived DNA segments as donors. A signalling function for transforming DNA also clarifies several general properties of specific transformation reactions.     

Regulation of cell division and malignant transformation

The problem of regulation of cell division is essentially a problem of understanding regulation of transition from the resting state of a cell to the dividing state and vice versa. In malignancy the ability to revert back to a normal resting state is impaired. A model is presented which attempts to explain the control of the above transitions through control of uptake of essential nutrients by a transport-inhibitory protein. Experimental evidence in favour of the model is given.     

病毒的细胞进入研究进展及其应用前景

病毒感染的初期事件包括病毒与细胞表面受体的相互作用和进入细胞的过程,而病毒的宿主细胞专一性很大程度上取决于这一阶段的专一识别特征和特殊要求。人乳头状瘤病毒、人免疫缺陷病毒和单纯疱疹病毒是感染人类的几种常见病原物,该文简要综述和讨论了与人体健康关系密切的这三种重要病毒表面的蛋白组分、宿主细胞表面受体及其相互作用和病毒的细胞进入的研究进展,以及在以病毒的细胞进入过程为靶点的抗病毒药物研发中的应用前景。     

Vanadium compounds promote the induction of morphological transformation of hamster embryo cells with no effect on gap junctional cell communication

Vanadium compounds were found to promote the induction of morphological transformation of hamster embryo cells. Exposure of the cells to Na−O-vanadate, vanadin (V) oxide or vanadin (IV) oxide sulfate following pre-exposure to a low concentration of benzo[a]pyrene, potentiated the induction of transformed colonies similar to 12-O-tetradecanoylphorbol-13-acetate. Unlike this phorbol ester, vanadium compounds did not inhibit intercellular communication, or activate protein kinase C. Nor did vanadate influence the reoccurrence of communication after removal of a communication blocking phorbol ester. On the other hand, vanadate showed strong synergism with the phorbol ester on induction of transformed morphology in the phorbol ester sensitive cell line BPNi. This suggests that vanadium and tumor promoting phorbol esters mediate their effect on the induction of morphological transformation of hamster embryo cells through different mechanisms.     

Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER

Intracellular antibodies (intrabodies) constitute a potent tool to neutralize the function of target proteins inside specific cell compartments (cytosol, nucleus, mitochondria and ER). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals and complements or replaces knockdown techniques such as antisense-RNA, RNAi and RNA aptamers. This article focuses on intrabodies targeted to the ER. Intracellular anti-bodies expressed and retained inside the ER (ER intrabodies) are shown to be highly efficient in blocking the translocation of secreted and cell surface molecules from the ER to the cell surface.The advantage of ER intrabodies over cytoplasmic intrabodies is that they are correctly folded and easier to select. A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins.The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells.In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing.In the future, endosomal localized receptors involved in bacterial and viral infections, intracellular oncogenic receptors and enzymes involved in glycosylation of tumour antigens might be new targets for ER intrabodies.     

Progression of spontaneous malignant transformation of epithelial rat liver cell lines

Cultured epithelial cell lines from normal rat livers were shown to undergo gradual transformation and malignancy which increased with time. Morphological changes appeared both before and after cells had attained a malignant state, as detected by agar tests. The progression of the degree of malignancy was determined by the morphological appearance of the cells, the increase in the number and size of cell colonies in soft agar, the expression of gamma glutamyl transferase (GT) and the shortening of the latency period necessary for tumor formation after transplantation to syngeneic rats of cells from sequential passages.     

Chemical carcinogen-mouse mammary tumor virus interactions in cell transformation

Summary We have studied the process of mammary cell transformation in vitro using a single cell clone (Clone 18) from a presumptive epithelial cell line, C57MG, derived from a normal mammary gland; a mouse mammary tumor virus (MMTV) host-range variant (RIII)vp4; and the potent initiating carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). After several serial subcultures, cells treated with virus and then with carcinogen exhibited an altered (transformed) morphology, a dramatic increase in anchorage independence, an increase in multinucleation after exposure to cytochalasin B, an enhanced ability to proliferate in low Ca²⁺ (0.01 mM) medium, and tumorigenicity when inoculated subcutaneously into athymic (nude) mice. Although some of these phenotypic alterations were observed also in cultures treated singly with MMTV or DMBA and in cultures exposed to DMBA before infection with MMTV, enhanced cytochalasin B multinucleation and tumorigenicity were properties observed only in mass cultures of cloned cells first infected with MMTV and then exposed to DMBA. This demonstrates for the first time that exposure of presumptive mammary epithelial cells to MMTV followed by DMBA, but not to either agent alone or to DMBA followed by MMTV, results in malignant transformation of these cells. Support for these studies was provided in part by the National Cancer Institute, Bethesda, Md., Contract N01-CP-01018.     

Effects of antimycotics on the biosynthesis of cellular macromolecules inAspergillus niger protoplasts

The effects of nine antimycotics on the biosynthesis of cellular macromolecules were analyzed using the regenerating system of protoplats ofAspergillus niger. The incorporation of several specific radioactive precursors into major cellular components were measured in the presence or in the absence of respective agents. Miconazole, ketoconazole, and tolnaftate inhibited the lipid synthesis. 5-Fluorocytosine strongly inhibited the DNA and protein syntheses. Griseofulvin, however, specifically inhibited the synthesis of cell wall polysaccharides, i.e. chitin and glucan. Other agents showed non-specific inhibition effects. The significance of morphological change of hypha as an indicator of antimycotic action and its feasibility as a screening tool for novel antimycotic compounds are discussed.Abbreviations GRP germination of regenerating protoplasts - TCA trichloroacetic acid     

The Croonian lecture 2006. Structure of the living cell

The smallest viable unit of life is a single cell. To understand life, we need to visualize the structure of the cell as well as all cellular components and their complexes. This is a formidable task that requires sophisticated tools. These have developed from the rudimentary early microscopes of 350 years ago to a toolbox that includes electron microscopes, synchrotrons, high magnetic fields and vast computing power. This lecture briefly reviews the development of biophysical tools and illustrates how they begin to unravel the 'molecular logic of the living state'.     

Changes in surface glycopeptides after malignant transformation of rat liver cells and during the regression of hepatoma cells

Normal liver cells, Zajdela's hepatoma cells, and regressing hepatoma cells were metabolically labeled with either radioactive glucosamine or mannose. Glycopeptides obtained by exhaustive pronase digestion of these cells were compared after fractionation by gel filtration on Bio-Gel P-6. Chemical analysis, affinity chromatography on immobilized lectins, alkaline treatment, and susceptibility toward endo-beta-N-acetylglucosaminidase and tunicamycin revealed dramatic changes in the glycopeptide patterns of transformed cells during the recovery of normal phenotype. The most prominent feature was the presence on the surface of hepatoma cells of a large glycopeptide, which was absent from normal liver cells and disappeared almost completely during the regression of hepatoma cells. This large glycopeptide had a Mr of 70,000, contained essentially O-glycosidically linked glycan chains, and did not result from a hypersialylation. N-glycosidically linked glycopeptides, high-mannose, and complex-type oligosaccharides were present in distinct proportions according to the differentiation state. Transformation of liver cells led to a reduction of high-mannose type oligosaccharides and an increase in the degree of branching of complex-type oligosaccharides. In addition, "bisected" glycopeptides were present only on hepatoma cells. The pattern of N-linked glycopeptides of normal liver cells was recovered during the regression of hepatoma cells. The origin of glycopeptide differences between normal and transformed cells and the evidence of a relation between carbohydrate changes, in particular the appearance of a large glycopeptide, and tumorigenicity are discussed.     

Convergent synthesis of neoglycopeptides by coupling of 2-bromoethyl glycosides to cysteine and homocysteine residues in T cell stimulating peptides

The 2-bromoethyl -glycosides of the disaccharide galabiose [Gal(1-4)Gal] and the trisaccharides globotriose [Gal(1-4)Gal(1-4)Glc] and 3-sialyllactose [Neu5Ac(2-3)Gal(1-4)Glc] have been prepared by improved routes. The 2-bromoethyl glycosides were then used in cesium carbonate promoted alkylations of the sulfhydryl groups of cysteine and homocysteine residues in T cell stimulating peptides. This convergent and general approach was used to prepare 16 neoglycopeptides which were obtained in 52–95% yields after purification by HPLC. 1H NMR spectroscopy revealed that -elimination and epimerization of neoglycopeptide stereocentres did not occur during the synthesis.