bluuebunny/crossref_metadata_2025 huggingface dataset:
Replication of the genome of alphaviruses
Authors: Kääriäinen Leevi , Takkinen Kristiina , Keränen Sirkka , Söderlund Hans
Published: May 2013

The genome of Semliki Forest virus (SFV) is 11 442 nucleotides with a 5′ cap-structure and a 3′ poly(A) tail of about 100 residues. The genome of the closely related Sindbis virus (SIN) is slightly longer (11 703 nucleotides). The parental RNA is first translated from the 5′ two thirds to yield; nsPl, nsP2, nsP3 and nsP4, which are cleaved from a polyprotein of 2431 amino acids (SFV). The parental genome is copied to a full-length minus strand with poly(U) at the 5′ end. The minus strand is used as template for the synthesis of 42 S RNA in membrane-bound replicative intermediate (RI) structures. In addition to 42 S RNA, a 3′-coterminal subgenomic 26 S mRNA, coding for the structural proteins, is synthesized by internal initiation at the minus strand. Capping and methylation of both plus-strand RNAs occur concomitantly with their synthesis.

Analysis of Sindbis virus temperature-sensitive RNA-negative mutants have shown that one complementation group (B) is specifically associated with the synthesis of minus strands. Another, group F, is involved in the polymerization step of both minus- and plus-strand 42 S RNA, and of the 26 S mRNA. The synthesis of minus strands is normally dependent on protein synthesis. There is a shut off of the minus-strand RNA synthesis at about 3h post-infection. This is apparently regulated by a virus-specific protein, represented by the complementation group A. The same protein is involved in the regulation of the initiation of 26 S RNA together with a component represented by group G mutants. Comparative analysis of SFV and SIN RNAs and DI RNAs of both viruses suggests that perhaps only 19 nucleotides from the 3′ end and about 150 nucleotides from the 5’ end are needed for replication of the alphavirus RNAs. In some SIN DI RNAs the proposed secondary structure at the 5’ end is replaced by a cellular tRNAASP suggesting that the secondary structure rather than nucleotide sequence is sufficient for the recognition by the viral polymerase.

Even when the primary structure of the four non-structural proteins of both SFV and SIN is known, the correlation of the genetic data with the individual proteins has not yet been possible.
Self-cleavage of rna in the replication of viroids and virusoids
Authors: Symons Robert H. , Hutchins Cheryl J. , Forster Anthony C. , Rathjen Peter D. , Keese Paul , Visvader Jane E.
Published: May 2013

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater-than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers.

We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both ‘plus’ and ‘minus’ RNA precursors during the replication cycle.

Similar self-cleavage of ‘plus’ RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.
The genome of herpes simplex virus: structure, replication and evolution
Authors: Mcgeoch Duncan J.
Published: May 2013

The objectives of this paper are to discuss the structure and genetic content of the genome of herpes simplex virus type 1 (HSV-1), the nature of virus DNA replicative processes, and aspects of the evolution of the virus DNA, in particular those bearing on DNA replication. We are in the late stages of determining the complete sequence of the DNA of HSV-1, which contains about 155 000 base pairs, and thus the treatment is primarily from a viewpoint of DNA sequence and organization.

The genome possesses around 75 genes, generally densely arranged and without long range ordering. Introns are present in only a few genes. Protein coding sequences have been predicted, and the functions of the proteins are being pursued by various means, including use of existing genetic and biochemical data, computer based analyses, expression of isolated genes and use of oligopeptide antisera. Many proteins are known to be virion structural components, or to have regulatory roles, or to function in synthesis of virus DNA. Many, however, still lack an assigned function.

Two classes of genetic entities necessary for virus DNA replication have been characterized: cisacting sequences, which include origins of replication and packaging signals, and genes encoding proteins involved in replication. Aside from enzymes of nucleotide metabolism, the latter include DNA polymerase, DNA binding proteins, and five species detected by genetic assays, but of presently unknown functions.

Complete genome sequences are now known for the related alphaherpesvirus varicella-zoster virus and for the very distinct gammaherpesvirus Epstein-Barr virus. Comparisons between the three sequences show various homologies, and also several types of divergence and rearrangement, and so allow models to be proposed for possible events in the evolution of present day herpesvirus genomes. Another aspect of genome evolution is seen in the wide range of overall base compositions found in present day herpesvirus DNAs. Finally, certain herpesvirus genes are homologous to nonherpesvirus genes, giving a glimpse of more remote relationships.
Activation of Keratinocyte Fibronectin Receptor Function During Cutaneous Wound Healing
Authors: Grinnell Frederick , Toda Ken-Ichi , Takashima Akira
Published: May 2013

Keratinocytes freshly isolated from unwounded skin could not attach and spread on fibronectin (FN)-coated culture dishes and could not bind and phagocytose FN-coated beads. These adhesive functions were activated, however, in Keratinocytes that were isolated from healing wounds. Moreover, adhesiveness of basal Keratinocytes to FN substrata was activated during epidermal cell or explant culture. Activation was specific for attachment to FN compared to other adhesion ligands, and occurred even when epidermal cells were cultured on collagen, basement membrane matrix, or lectin-coated substrata. Biochemical studies showed that Keratinocytes have a 140× 103Mr FN receptor analogous to the fibroblast receptor for FN, and that this receptor is expressed in activated Keratinocytes but not in Keratinocytes freshly isolated from unwounded skin. The absence of FN receptors from Keratinocytes in unwounded skin is not surprising since the basal Keratinocytes of the epidermis are attached to a basement membrane containing laminin and type IV collagen. During wound repair, however, these cells migrate over or through a FN-coated matrix. Consequently, expression of FN receptors may be an essential feature of healing. Believing that FN is the required substratum for keratinocyte migration during wound healing, we have initiated clinical studies to determine if topical application of FN is useful as a therapy for non-healing cutaneous ulcers.
Adhesive Interactions and the Metabolic Activity of Hepatocytes
Authors: Hughes R. Colin , Stamatoglou Stamatis C.
Published: May 2013

The nature and influence of adhesive interactions of rat hepatocytes with components of the extracellular matrix has been studied in culture. Hepatocytes interact with different kinetics to substrata composed of collagen type IV, laminin or fibronectin and adopt significantly different morphologies. The receptors mediating these various responses appear to be specific, according to the matrix, and in the case of fibronectin are complex, implicating several components of the hepatocyte surface. Collagen type IV maintains a differentiated phenotype more efficiently than fibronectin or laminin as measured by the production of adult hepatocyte markers such as albumin and repression of a-foetoprotein synthesis. Formation of matrix components is also influenced by the substratum: synthesis and secretion of fibronectin or collagen type IV is down-regulated when cells are cultured on the homologous substratum. Hepatocytes cultured in vitro secrete components of the coagulation cascade and also mediate fibrinolysis on addition of exogenous plasmin. The results are discussed in relation to the normal phenotype of the mature hepatocyte in vivo.
Influence of Cell Shape and Adhesiveness On Stratification and Terminal Differentiation of Human Keratinocytes in Culture
Authors: WATT FIONA M.
Published: May 2013

Human epidermal keratinocytes can be grown in culture under conditions in which they assemble a tissue with the same basic organization as normal epidermis. The cells stratify, mitosis is restricted to the basal layer and terminal differentiation occurs as the cells move through the suprabasal layers. Keratinocytes do not have to leave the basal layer in order to undergo terminal differentiation, but the two processes are normally linked, because during terminal differentiation the adhesive affinity of keratinocytes for the culture substratum and for other keratinocytes is reduced. Down-regulation of synthesis of basement membrane components and their receptors may provide the molecular basis for the reduction in cell-substratum adhesiveness. However, the molecules that mediate changes in cohesiveness have not yet been identified. Restriction of substratum contact, so that cells are prevented from spreading, appears to be one signal that induces keratinocytes to stop dividing and undergo terminal differentiation.
Studies on The Migration of Mouse Germ Cells
Authors: Donovan Peter J. , Stott David , Godin Isabelle , Heasman Janet , Wylie Christopher C.
Published: May 2013

Primordial germ cells in the mouse embryo migrate from their site of origin to the gonad where they differentiate, giving rise eventually to the gametes of the mature adult animal. The migratory phase is transient and therefore permits analysis of factors regulating the motile activity of cells in tissues. Germ cells can be isolated during migration and cultured on feeder cells of an established cell line (STO). In this system the molecular interactions mediating germ cell adhesion can be probed using antibodies to both germ cell surface determinants and to extracellular matrix components.
Analysis of In Vivo Cell Movement Using Transparent Tissue Systems
Authors: Thorogood Peter , Wood Andrew
Published: May 2013

The embryos of certain teleost species are transparent and cell behaviour within the intact embryo can be observed and recorded using Nomarski microscopy coupled with time-lapse video recording or time-lapse cine filming. In this report we review some of our recent analyses of cell behaviour patterns underlying key morphogenetic events. (1) Contact-guided cell migration through a structurally ordered extracellular matrix during fin development; (2) movement of tissue layers during epibolic overgrowth; and (3) cell ‘social’ behaviour during the establishment of the body axis (i.e. notochord formation and somitogenesis).

These results, on cell behaviour correlated with normal morphogenesis, provide a baseline for further work in which hypotheses concerning subcellular and molecular controls of cell behaviour can be tested by experimental perturbation in vivo.
Early Functional Differentiation in the Chick Embryonic Disc: Interaction between Mechanical Activity and Extracellular Matrix
Authors: Kučera Pavel , Monnet-Tschudi Florianne
Published: May 2013

The mechanical behaviour of ectodermal cells in the area opaca and the supracellular organization of fibronectin in the adjacent extracellular matrix were studied in whole chick blastoderms developing in vitro. The pattern of spontaneous mechanical activity and its modification by immunoglobulins against fibronectin were determined using a real-time image-analysis system. The pattern of fibronectin was studied using immunocytochemical techniques.

It was found that the ectodermal cells in the area opaca actively develop a radially oriented contraction, which leads to a distension of the area pellucida from which the embryo develops. Abnormally increased tension resulted in perturbations of gastrulation and neurulation. An optimized mechanical equilibrium within the blastoderm seems to be necessary for normal development. Anti-fibronectin antibodies applied to the basal side of the blastoderm led rapidly and reversibly to an increase of tension in the contracted cells. This observation indicates that modifications of the extracellular matrix can be transmitted to cytoskeletal elements within adjacent cells.

The extracellular matrix of the area opaca contains fibronectin arranged in radially oriented fibrils. This orientation corresponds to the direction of migration of the mesodermal cells. Interestingly, the radial pattern of fibronectin is found in the regions where the ectodermal cells are contracted and develop radially oriented forces. This observation suggests that the supracellular assembly of the extracellular materials could be influenced by the mechanical activity of adjacent cells. Possible modulations of the supracellular organization of extracellular matrix by other factors, e.g. diffusible metabolites, is also discussed.

The presence of characteristically organized extracellular matrix components, of spatially differentiated cell activities and of reciprocal interactions between them makes the young chick blastoderm an excellent system for physiological studies of the coordinated cellular activities that lead to changes in form, complexity and function.
Léucocyte Locomotion: Behavioural Mechanisms for Accumulation
Authors: Wilkinson P. C.
Published: May 2013

The behavioural mechanisms that may contribute to the accumulation of leucocytes (e.g. in inflammatory sites) are reviewed. Almost all of the neutrophils and monocytes of blood are motile and capable of migrating into tissues, but many blood lymphocytes are non-motile. They can however be induced to acquire locomotor capacity by culture with growth activators such as anti-CD3 antibodies. Resting lymphocytes become motile after entering the G1 phase of the cell cycle. Chemotaxis is well-studied in neutrophil leucocytes and is an efficient mechanism for cell accumulation. Possible mechanisms by which leucocytes respond to attractant gradients are discussed. Non-chemotactic mechanisms may also lead to cell accumulation, and evidence is reviewed that shows that an adhesive trapping mechanism causes leucocytes that cross an adhesion boundary to accumulate in the absence of a chemotactic gradient. Leucocytes migrating through aligned connective tissue show contact guidance in the axis of alignment of the tissue. Contact guidance can reinforce or hinder the response of neutrophils to chemotactic gradients.
Qualitative and Quantitative Analysis of Tumour Invasion in vivo and in vitro
Authors: Mareel Marc M. , Roy Frans M. Van , Messiaen Ludwine M. , Boghaert Erwin R. , Bruyneel Erik A.
Published: May 2013

Qualitative and quantitative methods for the analysis of invasion in ‘natural’ and in experimental tumours in vivo and in vitro are reviewed. In human tumours the functional consequences of invasion were evaluated histologically through staging on the basis of depths of invasion and through the presence of tumour cells inside vessels. Antibodies against components of the basement membrane have facilitated the definition of minimal invasion. With new probes derived from oncogene research the search for molecular differences between invasive and non-invasive parts of the tumour has begun. Since the same methods as those used for analysis of natural tumours also apply to experimental tumours in vivo, the major advantage of the latter is the possibility of manipulation. We have described a new mesenterium assay that may permit the selection of invasive cells from non-invasive ones in transfection experiments. Invasion relative to growth as a function of time was quantified in the kidney invasion test. In three-dimensional confrontations between embryonic chick heart fragments and invasive cells, we have used both a subjective grading and a qualitative computer-assisted image analysis of serial histological sections to score invasion. In two-dimensional confrontations supplementary methods could be applied, since such confrontations permitted direct observations on living cultures. In a variety of natural and experimental tumours, ultrastructural analysis, transmigration in two-compartment chambers, and release of metabolic label have demonstrated the role of motility and of lytic activity in tumour invasion.
Tumour suppressor genes
Authors: Klein George
Published: May 2013

Genes that can inhibit the expression of the tumorigenic phenotype have been detected by the fusion of normal and malignant cells, the phenotypic reversion of in vitro transformants, the induction of terminal differentiation of malignant cell lineages, the loss of ‘recessive cancer genes’, the discovery of regulatory sequences in the immediate vicinity of certain oncogenes, and the inhibition of tumour growth by normal cell products. Such tumour suppressor genes will probably turn out to be as, if not more, diversified as the oncogenes. Consideration of both kinds of genes may reveal common or interrelated functional properties.
Feedback regulators in normal and tumour tissues
Authors: Lord B. I.
Published: May 2013

Regulation of cell behaviour and population size is presumed to be not unlike classical regulation in non-biological systems, i.e. it is controlled by the cybernetic principle of negative feedback whereby the performance of progenitor cells depends inversely on a signal from their product, the size of which is proportional to the mass of the product. This signal may be inhibitory, acting directly on the progenitor cells. Alternatively, it may operate via an indirect and integrated inhibitor/stimulator feedback loop in which the one influences the production of the other. Illustrations taken from the various phases of haemopoietic development show the operation of these loops. Haemopoietic stem cells are under the direct influence of both inhibitor and stimulator but it is a feedback signal from the stem cell population that dictates the production of the one rather than the other. A second inhibitor acting at the stem cell level is a low molecular weight tetrapeptide which blocks the entry of cells into DNA synthesis, thus protecting them during a regimen of treatment with an S-phase cytotoxic drug. Proliferation of the maturing cells is also inhibited by feedback products of their fully mature descendants. Here, the effect is one of cell cycle modulation, whereas in the stem cell population the inhibitor and stimulator effect an on/off switch.

Attempts to characterize the molecules involved have been limited. A series of tri-to penta-peptides has been described for haemopoietic or epithelial cell inhibitors. A common feature of several is a pGlu-Glu end though whether this has any significance is not known.

In tumours it has been shown that some ascites are self-limiting and treatment of small tumours with cell-free fluid from a mature growth blocks their further growth. It appears that many tumour cells produce the feedback signals characteristic of their normal counterparts but are themselves less sensitive to it. The same is true of transforming growth factor-β which is produced and detected by virtually all cell types. In this case, the factor, inhibiting in most cases, is produced in inactive form and achieves its target specificity by a localized capacity to activate it. Some tumours, while responding to exogenous active TGF-/ β are incapable of activating the latent molecule.

It is concluded that the differential sensitivity of normal and neoplastic tissues to physiological feedback regulators is a potentially exploitable property in cancer therapy.
Regulatory factors of embryonic stem cells
Authors: Heath John K. , Smith Austin G.
Published: May 2013

The analysis of factors which regulate cell proliferation and differentiation in early mammalian development has been facilitated by the existence of cell lines derived from pluripotential stem cells of the early embryo; embryonal carcinoma (EC) cells and embryonic stem (ES) cells. EC cells have proved to be a useful source of embryonic growth factors. A potent mitogen, ECDGF has been isolated from EC cell conditioned medium. ECDGF appears to be a novel member of the heparin binding growth factor family. A remarkable feature of heparin binding growth factors is their ability to induce mesodermal differentiation in Xenopus laevis animal pole expiants. ES cell differentiation in vitro is controlled by exogenous factors, in particular a novel differentiation inhibitory factor DIA. Controlled bipotential differentiation of ES cells can be achieved by exposing ES cells to different combinations of regulatory signals.
Multi-lineage ‘stem’ cells in the mammalian embryo
Authors: Gardner R. L. , Beddington R. S. P.
Published: May 2013

The term ‘stem’ cell has acquired a rather more restricted meaning in cell biology than in embryology as a result of studies on the growth kinetics of renewing tissues in mature organisms. It is normally used in an embryological context as a synonym for ‘progenitor’ cell.

Methods of establishing the existence of multi-lineage progenitor cells in mammals are examined briefly before the occurrence and properties of such cells in both embryonic and extra-embryonic tissues of the mouse conceptus are reviewed. Various attributes of ‘stem’ cells that can be obtained from outgrowths of blastocysts in vitro are also discussed.
Early haemopoietic stem cells in the avian embryo
Authors: Dieterlen-Lievre Françoise , Pardanaud Luc , Yassine Fouzia , Cormier FranÇoise
Published: May 2013

Using ‘yolk sac chimaeras’, we have previously demonstrated that stem cells, destined to colonize haemopoietic organs other than the yolk sac, arise in the embryo proper. We have now investigated the emergence and potentialities of these cells in vivo and in vitro.

The in vivo approach consisted of interspecies grafting between quail and chick embryos. The cell progeny from the grafts was detected by means of QH1, a monoclonal antibody specific for the quail haemangioblastic lineage. When grafted into the dorsal mesentery of the chick embryo, which is a haemopoietic microenvironment, the region of the aorta from E3-E4 quail embryos generated large haemopoietic foci. When associated with a chick attractive thymic rudiment, cells left the quail aorta, entered this rudiment and underwent lymphopoiesis.

Cell suspensions prepared from 40-50 chick aortae, seeded in appropriate semi-solid media, yielded macrophage, granulocyte or erythrocyte clones. These colony forming cells were two to eight times more frequent than in cell preparations from hatchling bone marrow. By contrast, cells prepared from the whole embryonic body deprived of the aorta were not clonogenic.

By interspecies grafting of somatopleural (ectoderm + mesoderm, e.g. limb bud) or splanchnopleural rudiments (endoderm + mesoderm, e.g. lung, pancreas, intestine), the endothelial lining of blood vessels was shown to arise by two entirely different processes according to the rudiment considered: angiogenesis, i.e. invasion by extrinsic endothelial cells, in the limb bud, and vasculogenesis, i.e. in situ emergence of endothelial cells, in internal organs. The spleen, which first develops as a continuum to the pancreatic mesoderm, acquires its endothelial network by vasculogenesis, and is colonized by extrinsic haemopoietic stem cells. Granulopoietic cells in the pancreas and accessory cells in the lung are also extrinsic. Thus, in the case of endomesodermal rudiments, interspecies grafting reveals separate origins of endothelial and haemopoietic cells.
Identification of immunoglobulin heavy chain binding protein as glucose-regulated protein 78 on the basis of amino acid sequence, immunological cross-reactivity, and functional activity
Authors: Kozutsumi Yasunori , Normington Karl , Press Eileen , Slaughter Clive , Sambrook Joe , Gething Mary-Jane
Published: May 2013

Immunoglobulin heavy chain binding protein (BiP) associates transiently with various proteins destined for the secretory pathway. To investigate the relationship between BiP and the 78K (K=103Mr) glucose-regulated protein (GRP78), we have determined a partial amino acid sequence of purified mouse BiP and isolated and sequenced a full-length cDNA clone encoding mouse GRP78. The 26 amino-terminal residues of the mature BiP protein are identical to a sequence of amino acids located near the start of the open reading frame encoding GRP78. A polyclonal antiserum raised against mouse GRP78 protein expressed in bacteria from the cloned GRP78 cDNA could immunoprecipitate complexes consisting of BiP and unfolded forms of immunoglobulin heavy chains. Furthermore, a monoclonal antibody raised against mouse BiP immunoprecipitated mouse GRP78 expressed in monkey CV-1 cells from an SV40-GRP78 recombinant vector. Finally, like the endogenous BiP of simian cells, mouse GRP78 associated with malfolded, non-glycosylated forms of influenza hemagglutinin (HA) when GRP78 and HA were co-expressed from SV40 vectors in CV-1 cells. These studies confirm that BiP is identical to GRP78. Comparison of the nucleic acid and deduced amino acid sequence of mouse GRP78 with those of other rodent and human GRP78s revealed an extremely high degree of sequence identity. BiP/GRP78 is closely related (∼60% identity) to the cytoplasmic 70K heat-shock proteins. Surprisingly, the carboxy-terminal 29 amino acids of BÌP/GRP78, which are not conserved in HSP70 proteins, are almost identical in sequence to the steroidogenesis activator peptide found in the cytoplasm of rat Leydig tumor cells. Possible relationships between these polypeptides are discussed.
Import of proteins into the chloroplast lumen
Authors: Weisbeek Peter , Hageman Johan , Boer Douwe De , Pilon Rien , Smeekens Sjef
Published: May 2013

Plastocyanin is a nuclear-encoded protein that is functional in the thylakoid lumen of the chloroplast. It is synthesized in the cytoplasm as a precursor with an N-terminal transit peptide of 66 amino acids. Its transport route involves two steps, import into the chloroplasts and subsequent routing over the thylakoid membrane into the lumen. Concomitant with the transport, the transit peptide is removed in two successive steps. The transit peptide consists of two functionally different domains. In this study we examine to what extent each domain is involved in import and routing and how far these two processes are linked. For this purpose we made deletions in the N- terminal and C-terminal part of the transit peptide and fusion proteins which only contain one of these parts. The results show that the N-terminal part of the transit peptide is responsible for import into the chloroplast. The N-terminal 43 amino acids are sufficient to direct other proteins into the stroma. The C-terminal part of the transit peptide is a prerequisite for routing inside the chloroplast but not for import. When deletions are made in this part, the transport of plastocyanin stops after import and the intermediate accumulates in the stroma or on the outside of the thylakoids.

Transgenic tomato plants that constitutively express a foreign plastocyanin gene were used to study protein transport in different tissues. Normally, expression of endogenous plastocyanin genes in plants is restricted to photosynthetic tissues only. However, in the transgenic plants this foreign plastocyanin protein is found in all tissues examined. The protein is transported into the local plastids of these tissues and it is processed to the mature size. We conclude that plastids of developmentally different tissues are capable of importing precursor proteins that are normally not found in these tissues. Most likely such plastids, though functionally and morphologically differentiated, have similar or identical protein import mechanisms when compared to the chloroplasts in green tissue.

The precursor of ferredoxin was expressed in Escherichia coli. Surprisingly the precursor interacts with the cytoplasmic membrane and is translocated across this membrane. The unprocessed precursor accumulates in the periplasm.
Involvement of membrane lipids in protein export in Escherichia coli
Authors: Tommassen Jan , Vrije Truus de , Cock Hans De , Bosch Dirk , Kruijff Ben De
Published: May 2013

Several models for the transport of proteins across membranes predict a role for lipids. If these models are correct, then alterations in lipid metabolism may affect protein export and vice versa. We are investigating this possibility by studying Escherichia coli K-12 mutants with defects in protein export or phospholipid metabolism. A temperature-sensitive secA mutant, which is defective in protein export at 42°C, exhibited severe pleiotropic effects on membrane biogenesis. Incubation of this strain at 42°C resulted in the appearance of intracytoplasmic membranes, in alterations in lipopolysaccharide structure and in decreased cardiolipin and C18:1 fatty acid content. On the other hand, a pgsA mutant which is defective in the synthesis of acidic phospholipids, exhibited a protein export defect when studied in vivo or in vitro. These results are in agreement with a postulated role of membrane lipids in protein export.
Positioning of gene products during Caulobacter cell differentiation
Authors: Shapiro Lucy , Gober James W.
Published: May 2013

Caulobacter crescentus has one of the simplest known developmental programs that exhibits both temporal and spatial organization. A hallmark of the Caulobacter cell cycle is that the progeny cells that result from each cell division differ from one another with respect to structure and developmental program. The process of establishing asymmetry prior to cell division requires that a number of gene products be targeted to a pole of the predivisional cell and consequently segregated to one of the two progeny. Several products involved in flagellar biogenesis and the chemotaxis machinery are segregated to the swarmer cell. Evidence suggests that the protein product of somefla and che genes is targeted to the incipient swarmer cell pole. In the case of other flagellar genes, it is the mRNA that is apparently segregated to the swarmer cell. Two heat shock proteins, DnaK and Lon are specifically segregated to the progeny stalked cell.
Passage of Salmonella through polarized epithelial cells: role of the host and bacterium
Authors: Finlay B. Brett , Fry Jennifer , Rock Edwin P. , Falkow Stanley
Published: May 2013

Salmonella are intracellular parasites which enter their hosts by penetrating the intestinal epithelial barrier. We examined the interaction of .S’. choleraesuis and .S’. typhimurium with Madin Darby canine kidney (MDCK) and human larynx (HEp-2) epithelial cells to characterize bacterial adherence, invasion and penetration through epithelial monolayers. Epithelial cell microfilaments were required for bacterial internalization and surrounded the bacteria as they were internalized. The bacteria entered membrane-bound vacuoles inside epithelial cells where they replicated. When polarized MDCK cell monolayers were infected, we found that Salmonella could pass through this barrier and enter medium bathing the opposite surface, although most bacteria remained within the monolayer. Synthesis of several Salmonella proteins was induced by the presence of epithelial cell surfaces, and these proteins were required for bacterial adherence and invasion. This induction was stimulated by trypsin- and neuraminidase-sensitive structures on epithelial cells.
Regulation of the cell cycle timing of mitosis
Authors: Moreno Sergio , Hayles Jacqueline , Nurse Paul
Published: May 2013

Considerable advances have been made recently in our understanding of how the cell cycle timing of mitosis is regulated. This has come about because links have been established between two independent areas of research, one based on a genetic approach using the fission yeast Schizosaccharomyces pombe and the second based on a biochemical approach using Xenopus and starfish oocytes. In this chapter we review work that has identified a number of the mitotic regulating genes in fission yeast and has established links with controls operative in multicellular eukaryotes.
Further characterization of a size control gene in Saccharomyces cerevisiae
Authors: Cross Frederick R.
Published: May 2013

The DAF1-1 mutation reduces cell size and reduces or eliminates Gj phase in Saccharomyces cerevisiae, and results in a-factor resistance. DAF1-1 cells transferred into low cycloheximide express an increased G1 phase in their cycle, suggesting that Gj regulation is present but cryptic in the DAF1-1 cycle in rich medium. DAF1-1 reduces cell size by the criterion of RNA content per cell as well as cell volume. The α-factor resistance of DAF1-1 cannot be suppressed by bypassing the pheromone-receptor interaction with ‘signalling-constitutive’ mutations, suggesting that pheromone binding and initial signalling is normal in DAF1-1 strains, but that division arrest in response to the signal is specifically defective. Consistent with this idea, the cdc28–3 mutation significantly suppresses DAF1-1 α -factor resistance at permissive temperature; CDC28 is a gene required specifically for START and the G1/S transition, and does not affect pheromone response. Genetic results additional to those previously reported confirm that the wild-type dafl + /WHI1 gene is non-essential; this result may be surprising since the gene product is apparently ratelimiting for the G1/S transition: its deletion increases cell size, and multiple copies decrease cell size.
Control of G2 delay by the RAD9 gene of Saccharomyces cerevisiae
Authors: Weinert Ted , Hartwell Leland
Published: May 2013

In response to DNA damage, Saccharomyces cerevisiae cells arrest the cell cycle in the Gz phase. Arrest is defective in rad.9 mutants; rad9 cells divide and die without repairing the damage. Several cell cycle mutants that are defective in DNA replication arrest in G2 at the restrictive temperature; this arrest is due to the RAD? control function. Thus RAD? is responsible for the fact that mitosis is normally dependent upon DNA replication, a function we term a ‘checkpoint’. Four additional genes have been identified that are also components of the RAD9 checkpoint.
On the coupling between DNA replication and mitosis
Authors: Newport John , Dasso Mary
Published: May 2013

The rapid, early cell divisions in Xenopus laevis embryos are driven by an inflexible oscillator that is not influenced by the state of the DNA. In contrast, mitosis in somatic cells can be prevented by blocking replication or by damaging the DNA through irradiation. We have investigated the transition from the rapid, early cell cycle to the slower, more somatic-like cell cycle that occurs after division twelve in developingXenopus embryos, a stage called the mid-blastula transition (MBT). When aphidicolin, an inhibitor of DNA synthesis, was added to embryos just post-fertilization, the embryos continued to divide despite incomplete replication. Also, embryos incubated with aphidicolin from early times did not slow their cell cycles after division twelve as control embryos did, indicating a connection between the accumulation of DNA and the post-MBT timing of the cell cycle. However, incubation with hydroxyurea, an inhibitor of ribonucleotide reductase, resulted in an S phase arrest when the pools of dNTPs became depleted after division twelve. These experiments showed that the embryos had acquired the ability to arrest in S phase some time after the early divisions and before division thirteen. The acquisition of the ability to arrest in S phase did not depend upon new transcription. These experiments suggested that the number of nuclei present could be responsible for the extension of the cell cycle observed after the MBT. To investigate this, we added increasing concentrations of nuclei to an in vitro cell cycle system. We have shown that at high concentrations of nuclei the in vitro cycle is extended.
Analysis of the Cdc28 protein kinase complex by dosage suppression
Authors: Reed Steven I. , Hadwiger Jeffrey A. , Richardson Helena E. , Wittenberg Curt
Published: May 2013

In the interest of identifying components of the Cdc28 protein kinase complex, dosage suppression analysis was performed on temperature-sensitive and dominant negative CDC28 mutations. Dosage suppression is based on a rationale in which elevated expression of wild-type genes can rescue mutations in a target gene as a result of interaction between the respective encoded proteins. Three sequences capable of rescuing a temperature sensitive cdc28 mutation were isolated from a library of wild-type genomic DNA segments in the high copy vector YEpl3. Two of these, named CLN1 and CLN2 were found to encode closely related proteins with homology to cyclins. The third, CKSl, encodes an 18K (K=103Mr) protein that has been shown to be a component of the Cdc28 protein kinase complex and is a homolog of the sucl+ product of fission yeast. A number of dosage suppressors of the CDC28-dnl dominant negative mutation have been isolated. The one analyzed to date encodes a truncated subunit of the mitochondrial enzyme succinyl-CoA synthetase. The basis for suppression in this case remains to be elucidated.
M phase-promoting factor: its identification as the M phasespecific H1 histone kinase and its activation by dephosphorylation
Authors: Dorée Marcel , Labbé Jean-Claude , Picard André
Published: May 2013

A major protein kinase independent of Ca2+, cyclic nucleotide or diacylglycerol, the activity of which becomes maximal when cells enter M phase, decreases at ana-telophase, and is low during interphase, has been purified to near homogeneity from starfish oocytes and its catalytic subunit identified as p34cdc2. M phase-promoting factor (MPF) was found to co-purify with the M phasespecific kinase throughout its purification. p34cdc2 does not have to be associated with any specific protein for expression of Hl histone kinase or MPF activities. When p34’vfc2 is phosphorylated its protein kinase activity is inhibited, preventing entry into M phase, but once p34ede2 becomes dephosphorylated, its protein kinase activity increases and M phase is initiated. A second peak of MPF activity was separated from p34cdc2 in the ammonium sulfate fraction treated with ATP-y-S. It induced p34cde2 dephosphorylation and the concomitant stimulation of its kinase activity when injected in Xenopus or starfish oocytes.
Imaging and manipulation of cytosolic ions and messengers during cell activation
Authors: Harootunian Alec T. , Kao Joseph P. Y. , Adams Stephen R. , Tsien Roger Y.
Published: May 2013

Optical methods have recently become available for continuously imaging the free concentrations of important ions and second messengers such as calcium, sodium and hydrogen inside living cells. These ion levels are found to undergo remarkable changes upon stimulation of quiescent cells with growth factors known to stimulate phosphoinositide breakdown. In serum-starved REF-52 fibroblasts, growth factors such as serum, vasopressin, or PDGF (platelet-derived growth factor) cause intracellular [Na+] to increase from about 4 mM to 8 mM. If mitogen treatment is combined with pharmacological depolarization of the membrane potential, repetitive [Ca2+]i spikes result in these rat fibroblasts. The mechanism of this oscillation has been investigated by light-flash release of intracellular messengers such as inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), Ca2+, and diacylglycerol, as well as more traditional biochemical techniques. The key feedback pathway appears to be Ca2+-stimulation of phospholipase C production of Ins(1,4,5)P3.
Basic fibroblast growth factor (bFGF), a multifunctional growth factor for neuroectodermal cells
Authors: Westermann Reiner , Grothe Claudia , Unsicker Klaus
Published: May 2013

Basic fibroblast growth factor (bFGF), a heparin-binding mitogen for mesoderm-derived cells, also acts as a mitogen, differentiation inducing and maintenance factor for many neuroectodermal cells including glial cells, neurons, paraneurons, and their tumor counterparts. The molecule is expressed in several types of neuroectodermal cells in vitro and in vivo. Furthermore, bFGF occurs in many neuronal target tissues, and can prevent ontogenetic as well as lesion-induced neuron death. Thus, in terms of its wide range of functions, bFGF is apparently more than a ‘classical’ neurotrophic factor. Some of its essential features, such as regulation of expression, local availability and transport in the nervous system remain to be studied.
Growth factors as inducing agents in early Xenopus development
Authors: Slack J. M. W.
Published: May 2013

Factors from two growth factor families have been identified as having mesoderm inducing activity. These include activin and TGFβ from the TGFβ superfamily, and all members of the fibroblast growth factor (FGF) family.

When isolated ectoderm explants are treated with any of these factors, a proportion of their cells are caused to differentiate into mesodermal tissue types instead of epidermis. There are several differences in the biological activities which can broadly be summarized by saying that activin yields dorsal type inductions and FGF ventral type inductions.

Both bFGF and an FGF receptor have been detected in Xenopus blastulae, but it has not been shown that bFGF is normally secreted from vegetal cells. Various TGF/Mike mRNAs have also been detected and it is expected that an activin-like molecule will prove to be responsible for induction of the dorsal mesoderm in vivo.
Mechanisms of positional signalling in the developing eye of Drosophila studied by ectopic expression of Sevenless and rough
Authors: Hafen Ernst , Basler Konrad
Published: May 2013

In the developing eye of Drosophila cell fate is controlled by a cascade of inductive interactions. Little is known about how the specificity of positional signalling is achieved such that directly adjacent progenitor cells reproducibly choose distinct developmental pathways. The determination of the R7 photoreceptor in each ommatidium depends on the presence of the sevenless protein which acts as a receptor for positional information on the R7 precursor. The rough gene encodes a homeodomain protein that plays an instructive role in the determination of the R3 and R4 photoreceptor cells. The use of ectopic expression of sevenless and rough has provided insight into the mechanisms of positional signalling and the normal function of rough. Ubiquitous expression of sevenless does not alter cell fate suggesting that the inducing signal is both spatially and temporally controlled. Conversely, ectopic expression of rough in the R7 precursor causes a transformation of R7 cells into Rl-6 type cells. This indicates that rough acts, similar to other homeobox genes, as a selector gene that determines the fate of single cells.
The biochemistry and biology of the myeloid haemopoietic cell growth factors
Authors: Heyworth C. M. , Vallance S. J. , Whetton A. D. , Dexter T. M.
Published: May 2013

In the adult, blood cell production or haemopoiesis takes place mainly in the bone marrow. The blood cell types produced are a reflection of the needs of the organism at any moment, for example bacterial infection leads to a large increase in neutrophil production. The rate and scale of blood cell production in vivo are regulated, at least in part, by the synthesis and release of specific cytokines both within the bone marrow and also from other tissues. Here we detail the range of cytokines which act directly on haemopoietic stem cells and myeloid progenitor cells. Also cellular systems which will permit the elucidation of the specific interactions between these various cytokines which regulate stem cell self-renewal, differentiation and proliferation are described.
Growth and differentiation factors of pluripotential stem cells
Authors: Heath John K. , Smith Austin G. , Hsu Li-Wei , Rathjen Peter D.
Published: May 2013

The mammalian embryo develops as a quasi-stem cell system whose differentiation and pluripotentiality in vitro is controlled by a single regulatory factor, Differentiation Inhibiting Activity/Leukemia Inhibitory Factor (DIA/LIF). DIA/LIF is expressed in two distinct functional forms, derived from the use of alternate transcriptional start sites, one of which is freely diffusible and the other tightly associated with the extracellular matrix. The dissemination of the DIA/LIF signal is therefore under specific molecular control. The expression of DIA/LIF in vitro is both developmentally programmed and controlled by the action of other growth factors, the most notable of which are members of the fibroblast growth factor family expressed by the stem cells themselves. This indicates that differentiation and proliferation in early development of the mouse are controlled, at least in part, by an interactive network of specific growth and differentiation regulatory factors.
Characterization of INT-2: A member of the fibroblast growth factor family
Authors: Dickson Clive , Acland Piers , Smith Rosalind , Dixon Mark , Deed Richard , Macallan David , Walther Wolfgang , Fuller-Pace Frances , Kiefer Paul , Peters Gordon
Published: May 2013

int-2 was discovered as a proto-oncogene transcriptionally activated by MMTV proviral insertion during mammary tumorigenesis in the mouse. Sequence analysis showed int-2 to be a member of the fibroblast growth factor family of genes. In normal breast and most other adult mouse tissues, int-2 expression was not detected except for low levels in brain and testis. However, using in situ hybridization, expression was found at a number of sites during embryonic development, from day 7 until birth. An analysis of the int-2 transcripts found in embryonal carcinoma cells revealed six major classes of RNA initiating at three promoters and terminating at either of two polyadenylation sites. Despite the transcriptional complexities, all size classes of RNA encompass the same open reading frame. Using an SV40 early promoter to drive transcription of an int-2 cDNA in COS-1 cells, several proteins were observed. These were shown to be generated by initiation from either of two codons: One, a CUG, leads to a product which localizes extensively to the cell nucleus and partially to the secretory pathway. In contrast, initiation at a downstream AUG codon results in quantitative translocation across the endoplasmic reticulum and the accumulation of products ranging in size from 27.5×103Mr to 31.5×103Mr in organelles of the secretory pathway. These proteins represented glycosylated and non-glycosylated forms of the same primary product with or without the signal peptide removed. These findings suggest the potential for a dual role of int-2-, an autocrine function acting at the cell nucleus, and a possible paracrine action through a secreted product.