bluuebunny/crossref_metadata_2025 huggingface dataset:
Vesicle Movements And Microtubule-Based Motors
Authors: P. Sheetz Michael , Vale Ronald , Schnapp Bruce , Schroer Trina , Reese Thomas
Published: May 2013

The movements of many cytoplasmic vesicles follow the paths of microtubules, some moving in one direction and others moving in the opposite direction on the same microtubule. Recently we have isolated one cytoplasmic motor, kinesin, and defined another, the axoplasmic retrograde factor, both of which are capable of powering anionic latex beads in both directions along polar microtubule arrays. Evidence summarized here supports but does not prove the hypothesis that kinesin and the retrograde motors are indeed responsible for powering vesicle movements.
Characterization Of The Sea-Urchin Egg Microtubule-Activated Atpase
Authors: A. Collins Christine , B. Vallee Richard
Published: May 2013

We have found that cytoplasmic extracts from unfertilized sea-urchin eggs contain a prominent microtubule-activated ATPase activity. This activity is induced by polymeric tubulin, but not by tubulin subunits. The activity cosediments with taxol-stabilized microtubules in an ATP- independent manner. We have separated the ATPase from cytoplasmic dynein and other ATPases on sucrose gradients. The sedimentation, enzymic and microtubule-binding properties of the microtubule-activated species show it to be distinct from cytoplasmic dynein, myosin and kinesin. Since the major function of microtubules in the early sea-urchin embryo is in mitosis, this enzyme represents a new candidate for a role in spindle motility.
Cytoskeletal Dynamics In The Mouse Egg
Authors: Maro Bernard , Howlett Sarah K. , Houliston Evelyn
Published: May 2013

The distribution and roles of the microtubule and microfilament networks in the mouse egg following fertilization are described. The role of the chromosomes in the control of the egg cytoskeleton organization is discussed and a model for polar body formation proposed. Finally we describe the changes occurring in the pattern of proteins synthesized during this period, these being discussed in relation to cell cycle events and to changes in cytoskeleton organization.
The Role Of Microtubule Polarity In The Movement Of Kinesin And Kinetochores
Authors: J. Mitchison T.
Published: May 2013

Microtubules are important for organizing and directing many types of intracellular motility. Recently progress has been made in the analysis of two types of motility at the molecular level: the movement of axonal vesicles driven by kinesin, and the movement of chromosomes driven by the kinetochore. Both require ATP for movement in vitro. Kinesin-driven movement is unidirectional, towards the microtubule plus end, while movement of the kinetochore is bidirectional. These similarities and differences are discussed and incorporated into a new model for the kinetochore- microtubule interface.
Reticulomyxa: A New Model System Of Intracellular Transport
Authors: Koonce Michael P. , Euteneuer Ursula , Schliwa Manfred
Published: May 2013

Reticulomyxa is a large multinucleated freshwater protozoan that provides a new model system in which to study intracellular transport and cytoskeletal dynamics. Within the cell body and reticulopodial network, rapid, visually striking saltatory organelle motility as well as bulk cytoplasmic streaming can be readily observed. In addition, the cytoskeletal elements within these strands undergo dynamic splaying and fusing rearrangements, which can be visualized by videoenhanced light microscopy. A reactivatable lysed cell model has been developed that appears to preserve, and therefore permits examination of, these three forms of motility in a more controlled environment. Individual organelle movements are microtubule-based and have similarities to, but also differences from, the recently described kinesin-based transport. This lysed cell model can be further manipulated to provide native, ordered, completely exposed networks of either microtubules or microfilaments, or a combination of both, and thus may serve as a versatile motility assay system in which to examine the movement of exogenously added isolated organelles or latex beads.
Retrovirus genome replication: priming specificities of plus-strand dna synthesis
Authors: Taylor John , Sharmeen Lamia
Published: May 2013

The retroviral reverse transcriptase is a multifunctional protein. Not only does it contain both RNA- and DNA-directed DNA synthesis activities but also it contains an endonuclease activity necessary for the integration of viral RNA and a RNase H. This latter activity can reduce to oligoribonucleotides viral RNA that has been reverse transcribed into minus-strand DNA. However, during avian retrovirus genome replication it does this in a highly specific manner so as to generate a specific 12-base primer for plus-strand DNA synthesis. Even though many other oligoribonucleotides are also made there is an efficient selection xof the specific primer followed by its efficient utilization in plus-strand DNA synthesis, and subsequent removal. We have used a reconstructed system to gain an understanding of the factors that contribute towards these observed specificities.
Dna and protein interactions in the regulation of plasmid replication
Authors: Filutowicz Marcin , Mceachern Michael J. , Mukhopadhyay Pradip , Greener Alan , Yang Shengli , Helinski Donald R.
Published: May 2013

As for bacterial and animal viruses that employ different mechanisms for their duplication in a host cell, plasmids have evolved different strategies to assure their hereditary stability or maintenance at a specific copy number during cell growth and division. A characteristic feature of plasmid replication control, however, is an involvement of one or more negatively controlling elements. Furthermore, a majority of the bacterial plasmids examined to date contain direct nucleotide sequence repeats at their origin of replication and encode a replication protein that binds to these repeat sequences. The binding of the replication protein (n protein) specified by the antibiotic resistance plasmid R6K to a set of 22 base pair direct nucleotide sequence repeats is required for the initiation of replication at each of three origins of replication (α, βand γ) within a 4 Kb segment of R6K. The π initiation protein is multifunctional in that it has both positive and negative activities in both controlling the initiation of replication and autoregulating its own synthesis. Similarly, the direct repeats of plasmid R6K and several other plasmid systems play more than one role in plasmid replication. These repeats, termed iterons, are not only required for origin activity but also exert a negative effect on plasmid copy number possibly as a result of their ‘titration’ of a plasmid encoded replication protein. The properties of plasmid replication proteins and direct nucleotide sequence repeats that are important for their opposing positive and negative roles in the regulation of the initiation of replication are described with particular emphasis on plasmid R6K of Escherichia coli.
Actin Cortex and Microtubular System in Morphogenesis: Cooperation and Competition
Authors: Vasiliev J. M.
Published: May 2013

Actin cortex and microtubules determine two different types of morphological organization of the cytoplasm. Cooperation and competition between these two organizations may produce a diversity of final forms. Actin cortex alone, without the other cytoskeletal systems, is sufficient for the organization of vectorized pseudopod-forming cytoplasmatic units termed ‘actinoplasts’. Reversible segregation of cytoplasm into actinoplasts and microtubule-rich stable domains (‘tubuloplasts’) is suggested to play an important role in many types of morphogenesis. Segregation of this type can be induced in fibroblasts of certain lines by the tumour promotor, tetradecanovl phorbol-acetate (TPA). Self-organization of each actinoplast leads to the development of microfilament bundles associated with focal contacts. Analysis of the evolution of these bundle-contact structures during spreading of mouse fibroblasts suggests that their patterns are determined by the degree of centripetal tension within the actin cortex; the microtubular system stabilizes these patterns. Pseudopodia! activity of the edges of the actinoplasts can be controlled by environmental factors. One particular type of control, i.e. the effect of cell-cell contact, is discussed. Actin cortex and microtubules seem to have alternative effects on the distribution of vimentin-containing intermediate filaments.
Adhesion Plaques: Sites of Transmembrane Interaction Between the Extracellular Matrix and the Actin Cytoskeleton
Authors: Burridge Keith , Molony Leslie , Kelly Thomas
Published: May 2013

In this paper we review what is known about the organization of adhesion plaques, the regions where cells in culture adhere most tightly to the underlying substratum. These specialized areas of the plasma membrane serve as attachment sites for stress fibres. A major objective has been to determine how microfilament bundles are anchored at such regions. In their morphology and composition adhesion plaques resemble the adhesions fibroblasts make to the extracellular matrix. Some extracellular matrix components have been identified on the outside face of adhesion plaques. Within the plasma membrane of adhesion plaques, extracellular matrix receptors, such as the fibronectin receptor (integrin), have been identified. This transmembrane glycoprotein complex has been shown to bind the cytoplasmic protein talin, which, in turn, associates with vinculin. These proteins establish a transmembrane chain of attachment between the extracellular matrix and the cytoskeleton, although how the actin filaments interact with these components remains to be determined. Besides having a structural function, adhesion plaques may also be regions where regulatory signals are transmitted across the membrane. Consistent with this idea has been the finding that various tyrosine kinases and a calcium-dependent protease are concentrated at the cytoplasmic aspect of adhesion plaques. Furthermore, several adhesion plaque proteins become phosphorylated during cell transformation by Rous sarcoma virus. In future work it will be important to determine how such modifications affect the interactions of these proteins and the stability of adhesion plaques.
Molecular Interactions in Adherens-Type Contacts
Authors: Geiger Benjamin , Volk Talila , Volberg Tova , Bendori Ronit
Published: May 2013

Adherens junctions are members of a molecularly and structurally heterogeneous family of cell contacts sharing a common association with the microfilament system. Various topics related to the biogenesis of these cellular contacts and the molecular interactions involved in their formation are discussed. ⅓ The role of vinculin, a cytoplasmic ‘plaque’ component present in all adherens junctions tested to date and its possible interactions with the other junctional domains have been investigated by both biochemical analyses and studies of molecular dynamics in microinjected living cells. The importance of A-CAM, which apparently functions as a ‘junctional receptor’ is emphasized and its roles in junction formation in cell cultures and in developing embryos are discussed. In addition, its relationship to other Caz+-dependent cell adhesion molecules (in particular L-CAM) is considered. The evidence indicating that the level of expression of vinculin-specific mRNA is affected by culture conditions and may be markedly modulated by changes in the adhesiveness of the substratum on which the cells grow is reviewed.
Fluidity of the Neural Epithelium During Forebrain Formation in Rat Embryos
Authors: Morriss-Kay Gillian , Tuckett Fiona
Published: May 2013

During neurulation in rat embryos, the forebrain grows more rapidly than can be accounted for by intrinsic cell division alone, while the adjacent midbrain/rostral hindbrain maintains a constant cell number despite a high mitotic index (the cell cycle time is 6h throughout the neural epithelium). We have proposed that neuroepithelial cells flow in a rostral direction within the midbrain/rostral hindbrain region, towards and into the rapidly expanding forebrain. Evidence in support of this hypothesis is provided by cell-labelling studies: labelled neuroepithelial cells were injected into specific sites in the midbrain or rostral hindbrain neuroepithelium of unlabelled embryos ; after culture of these embryos for 24 h, the labelled cells were found in positions rostral to the injection sites.

In the midbrain/rostral hindbrain region, mitotic spindles were found to be predominantly orientated parallel with the long axis of the embryo; transversely orientated spindles were more frequently observed close to the lateral edges than more medially. Neural crest cells emigrate from the lateral edges during neurulation. These observations suggest that mitotic spindle orientation reflects the direction of cell movement: in the lateral region movement towards the lateral edge would maintain cell number in the transverse plane as neural crest cells emigrate; elsewhere, cells are moving mainly in the longitudinal plane, towards the forebrain. The possible causal relationship between cell movement and mitotic spindle orientation is discussed.

Cell flow within the intact neural epithelium is compared with cell flow in the intestinal villus epithelium. Other types of epithelial cell movement observed in the cranial neural epithelium during neurulation include expansion and shrinkage of the epithelial surface associated with change of cell shape and microfilament-mediated curvature. Cell rearrangement involving exchange of neighbours and cell movement towards and into a site of epithelio-mesenchymal conversion are also implicated.
A Unified Approach to Analysing Cell Motility
Authors: Dunn G. A. , Brown A. F.
Published: May 2013

The quantitative analysis of cell motility in culture has several important functions. First, it gives a concise and accurate description of the motile process and can detect subtle differences in motility- due to different genetic makeup or experimental conditions. Second, its objectivity means that results can be communicated precisely and used unambiguously to test hypotheses about motility. Third, it may be used to derive a mathematical model with the same statistical properties as the motile process and thus elucidate the mechanism of motility.

In this paper, we introduce a general procedure for analysing cell motility in a wide variety of circumstances. We describe a pilot project for the analysis of simple geometrical data obtained from randomly moving fibroblasts. Finally, as an example, we show how an analysis of the translocation of the fibroblasts can lead to insights into the mechanism of motility that are arguably not obtainable by any other approach.
Cell Motility and the Problem of Anatomical Homeostasis
Authors: Harris Albert K.
Published: May 2013

The locomotion of tissue cells need not be invasive or disrupt normal tissue geometry, as occurs in cancer. The normal relationship between anatomical structure and cell locomotion is exactly the reverse, with motility serving to create and maintain the structures of the body. This relationship is most extreme in sponges, where time-lapse films show that the cells move about continually in patterns that restructure these animals’ simple anatomy. The cells choose their position according to their differentiated cell type, which is the opposite of what is usually assumed to occur in development and has important implications for the functional significance of histotypic cell sorting.

A particular type of cellular force that seems to be important in morphogenesis is the traction that all motile tissue cells exert. This traction can be studied by culturing cells on very thin sheets of silicone rubber, so that the locations and variations in the cellular forces are made visible by the wrinkles they produce in the rubber substratum. One finding has been that the traction forces exerted by untransformed fibroblasts are very much stronger than is needed for their own locomotion, but are well adapted for the function of rearranging and aligning collagen fibres to form structures like ligaments, tendons and muscles. These forces are found to be greatly weakened by neoplastic transformation, however, suggesting that malignant invasiveness results from some sort of deflection of cell traction forces from their proper morphogenetic functions, so as to produce uncontrolled invasion.

To explain how motile cells create and maintain structures, as well as how their locomotion sometimes becomes perverted into the form of cancerous invasiveness, what seems to be needed is an extension of the concept of homeostasis to apply to the control of geometric relations between cells. This task may not be easy; one obstacle is the widespread belief that asymptotic stability implies the minimization of free energy. Instead, I suggest that stability results from balance of opposing forces within tissues, and that genes control which anatomical shapes will exist by determining the rules by which the relative strengths of these forces vary as functions of shape: to control shape, one must control the way forces vary with shape.
Recombinant retrovirus-mediated gene transfer to normal haemopoietic cells
Authors: Johnson G. R.
Published: May 2013

Several of parameters seeking to increase the infection frequency of normal haemopoietic cells by recombinant retroviruses containing a drug selectable marker and/or a haemopoietic growth factor gene have been compared. By using bone marrow cells pretreated with 5-fluorouracil, cocultivation of cells for at least 4 days with virus producing fibroblasts, and addition of pokeweed mitogen stimulated spleen cell conditioned medium, from 30 to 100% of haemopoietic spleen colony forming cells (CFU-S) could be infected.

Because drug (G-418) selection was found to select subsets of CFU-S, a retrovirus lacking the drug selectable marker Neor, but containing a GM-CSF gene, was used to study the effects of endogenous GM-CSF production on CFU-S differentiation. Infected CFU-S produced equivalent numbers of erythroid and neutrophil-macrophage committed progenitor cells to control uninfected CFU-S. However, the progeny of infected CFU-S produced GM-CSF, some cells grew autonomously and neutrophil numbers were greatly increased at the expense of erythroblasts. These results suggest that unregulated GM-CSF production, although not altering commitment, alters the amplication phase following commitment.
The ras oncogenes
Authors: Marshall C. J.
Published: May 2013

Oncogenic forms of the p21ras genes have been found in a large variety of human malignancies and tumours induced in animals by chemical carcinogens or irradiation. The active form of the p21 ras proteins is the GTP bound state and oncogenic mutations result in the protein being constitutively in the GTP bound active state. There is evidence to suggest that activating mutations can occur either as initiating steps in carcinogenesis or as later events in the evolution to frank neoplasia. To transduce a signal for proliferation and transformation the active GTP form of p21ras must interact with one or more cellular targets. Genetic experiments suggest that one potential effector molecule is the GTPase activating protein GAP. However, the mechanism by which interaction with GAP results in proliferation and transformation remains to be elucidated.
Mesenchymal influences on epithelial differentiation in developing systems
Authors: Sharpe P. M. , Ferguson M. W. J.
Published: May 2013

Mesenchyme tissue: cells, matrix and soluble factors, influence the morphogenesis, proliferation and differentiation of a variety of embryonic epithelia, e.g. in the tooth, skin, mammary and salivary glands. Mesenchyme derivatives also ‘maintain’ adult epithelia, e.g. the local proliferation rate and cytokeratin composition of oral mucosa. Abnormalities in such epithelial-mesenchymal interactions lead to a variety of pathologies such as premalignant lesions, e.g. leukoplakia, tumours and psoriasis, whilst therapeutic manipulation of such interactions can prevent the exfoliation of dental implants. In all of these systems it is critical to understand, at the cellular and molecular levels, how the mesenchyme signals to the epithelium and how the latter processes and responds to such signals. We have investigated such questions using the developing embryonic palate both as a model system and as an important organ: failure of mesenchymal signalling leads to the common and distressing birth defect of cleft palate.

Bilateral palatal shelves arise from the maxillary processes of embryonic day 11 (Ell) mice, grow initially vertically down the sides of the tongue, elevate on E13-8 to a horizontal position above the dorsum of the tongue and fuse with each other in the midline on E14. The medial edge epithelia of each shelf fuse with each other to form a midline epithelial seam, suprabasal cells die, and the basal (stem) cells synthesize extracellular matrix molecules and turn into mesenchymal cells. Simultaneously the oral epithelia differentiate into stratified squamous cells and the nasal epithelia into pseudostratified ciliated columnar cells. Oral, medial and nasal epithelial differentiation is specified by the underlying mesenchyme in vivo and in vitro. Signalling involves a bifurcating action of a combination of soluble growth factors e.g. TGF-α, TGF-β, PDGF and FGF on palatal epithelia and mesenchyme. These factors stimulate the synthesis of specific extracellular matrix molecules by palate mesenchyme cells, and the appearance of receptors for such molecules on epithelial cells. In this way, a combination of mesenchymal soluble factors and extracellular matrix molecules direct palatal epithelial differentiation. These signals act on epithelial basal (stem) cells, causing them to synthesize unique proteins, which may direct subsequent differentiation of daughter cells. In the most extreme example, namely the medial edge epithelia, these signals result in the basal epithelial cells transforming into mesenchymal cells, thus demonstrating that they are indeed multipotential stem cells.
Stem cell renewal and differentiation in acute myeloblastic leukaemia
Authors: Mcculloch E. A. , Minden M. D. , Miyauchi J. , Kelleher C. A. , Wang C.
Published: May 2013

The defining properties of stem cells are capacities for self-renewal and, after determination, a limited number of terminal divisions. The blast cells of acute myeloblastic leukaemia (AML) are maintained by stem cells with these two properties. Since renewal and differentiation can be assessed separately in cultures of AML blasts, these cancer cells provide a useful model for examining stem regulation; such studies have practical importance for future developments in the treatment of AML. This paper considers three aspects of blast cell biology. First, evidence is presented that self-renewal and differentiation are regulated by specific genes; further, the DNA encoding these genes has structural features that affect the chemosensitivity of self-renewal. This sensitivity varies from patient-to-patient and is an important attribute contributing to variation in treatment efficacy. Second, the effects of myelopoietic growth factors on blast stem cells are presented and discussed, as these bear on the regulation of the balance between renewal and differentiation. Finally, models of leukaemic haemopoiesis are considered in light of the experimental findings. The suggestion is advanced that leukaemia can be explained better by abnormalities of gene expression than by blocked differentiation.
Stem cells in mammary gland differentiation and cancer
Authors: Rudland Philip S. , Barraclough Roger
Published: May 2013

Evidence based on ultrastructure and immunocytochemical staining suggests that morphological gradations between epithelial and myoepithelial cells, and possibly between epithelial and alveolar-like cells, can occur in terminal ductal structures of rat and human mammary glands. In neoplastic disease the benign, carcinogen-induced rat and benign, human mammary tumours can contain epithelial, myoepithelial-like and alveolar-like cells, whereas their malignant counterparts mainly contain only epithelial-like cells. Clonal epithelial cell lines from normal rat mammary glands, from benign tumours and from SV4O-transformed human mammary cultures can differentiate to either myoepithelial-like or alveolar-like cells. In those of the rat, the differentiation processes occur in steps, intermediate cells along the myoepithelial-like pathway resemble the morphological intermediates in the terminal ductal structures in vivo. Changes in specific polypeptides characterize each of the intermediate cells in vitro. One of the earliest increases observed in the myoepithelial-like pathway in vitro is that due to a novel protein p9Ka, whereas the major increases in Thy-1 antigen and the basement membrane proteins laminin and type IV collagen occur at later steps. The nucleotide sequence of the gene for p9Ka is related to that of the small, regulatory calcium-binding proteins, and antibodies raised to synthetic fragments of its predicted amino acid sequence react with only myoepithelial cells within the rat mammary parenchyma. Increases in the production of p9Ka and Thy-1 are largely due to increases in their messenger RNAs, possibly arising at the level of transcription of the DNA, whereas the increases in production of laminin and type IV collagen occur at a post-transcriptional level. The normal transcriptional promoter sequences of TATA or CAAT are not found adjacent to the genes for p9Ka or Thy-1. Cells and cell lines from malignant rat mammary tumours of increasing metastatic potential and from malignant areas of human ductal carcinomas largely fail to yield fully differentiated myoepithelial-like or alveolar-like cells in culture; however, weakly metastasizing rat cells yield variants which may retain a vestige of the myoepithelial phenotype. It is suggested that novel regulatory transcriptional element(s) may control the production of some of the proteins along the normal myoepithelial-like pathway, and that these elements may be relatively unique in their capacity to become inoperative in the malignant breast cancer cell.
Differentiation of fibroblast stem cells
Authors: Bayreuther Klaus , Rodemann H. Peter , Francz Pal. I. , Maier Katharina
Published: May 2013

Primary human skin fibroblasts derived from the abdomen of 45 female donors of the four age groups 1–20, 20–40, 40–60, and 60–80 years were studied in primary explant, in primary low-density mass cultures, and in primary clonal populations in vitro. As a function of the age of the donor, primary mitotic and postmitotic fibroblasts in the three primary cell systems analysed represent heterogeneous populations with reproducible changes in the proportions of the mitotic fibroblasts MF I, MF II, MF III, and postmitotic fibroblasts PMF IV, PMF V, PMF VI, and PMF VII. These findings make it very likely that equivalent cell types exist in the connective tissue of skin in vivo, and that these cells undergo reproducible changes in the proportions of the mitotic and postmitotic counterparts in vivo as a function of the age of the donor. Secondary mitotic human skin fibroblast populations of the cell line HH-8 in vitro underwent 53-6 ± 6-0 cumulative population doublings (CPD) in 302 ± 27 days. If appropriate methods are applied, mitotic fibroblasts differentiate spontaneously into postmitotic fibroblasts which are kept in stationary cultures for up to 305 ± 41 additional days.(As a function of the CPD level and of the duration of stationary culture, secondary mitotic and postmitotic fibroblast populations are heterogeneous populations with reproducible changes in the proportions of mitotic fibroblasts MF I, MF II, and MF III, and postmitotic fibroblasts PMF IV, PMF V, PMF VI, and PMF VII. The seven secondary fibroblast cell types show differentiation-dependent and cell-type specific patterns of [35S]methionine polypeptides in total soluble cytoplasmic and nuclear proteins, in secreted proteins, and in membrane bound proteins. These findings make it very likely that the morphologically recognizable primary and secondary fibroblasts differentiate spontaneously along a seven stage terminal cell lineage MF I - MF II - MF III - PMF IV - PMF V - PMF VI - PMF VII in three compartments of the fibroblast stem cell system.
A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium
Authors: Gray Lindsay , Baker Karen , Kenny Brendan , Mackman Nigel , Haigh Richard , Holland I. Barry
Published: May 2013

Escherichia coli haemolysin (HlyA), a 107K (K = 103Mr) protein, is secreted to the medium in an hlyB, hlyD-dependent process. Secretion, however, depends on neither an N-terminal signal sequence nor on SecA, which is part of the normal cellular export machinery for periplasmic and outer membrane proteins. In contrast, HlyA contains a novel C-terminal secretion signal encompassing the last 27 amino acids and possibly some additional residues immediately upstream. This region is characterized by a 16 residue ‘aspartic acid box’ composed largely of small amino acids which we propose constitutes an important element in recognition of the membrane translocation complex constituted by HlyB and HlyD. This feature is also found at the C-terminus of the adenyl cyclase and leukotoxin A molecules and resembles a recently identified eukaryotic C-terminal signal for targeting to glycosomes.

A domain of the HlyB component of the haemolysin transport system is also similar to a domain widely distributed in nature, apparently acting as an ATP-dependent transport protein for a wide variety of molecules. Secretion of haemolysin, however, is the first example of a protein translocation system involving an HlyB-like molecule. This suggests that a major role of HlyB or at least its C-terminal domain is the coupling of energy to translocation of the haemolysin. It is more likely therefore that HlyD is more involved in the actual translocation through the membrane.

On the basis of genetical and biochemical studies we propose that the haemolysin is translocated directly to the medium bypassing the periplasm. We further propose that HlyB and HlyD together constitute a membrane-bound translocator specific for molecules bearing the HlyA targeting sequence, and that the organization of this complex (conceivably involving other E. coli membrane proteins) must somehow straddle the inner and outer membranes.

Finally, the HlyA C-terminal domain has been successfully used to promote the secretion to the medium of a number of heterologous polypeptides, in an HlyB,D-dependent manner.
The use of gene-fusions to determine membrane protein topology in Saccharomyces cerevisiae
Authors: Green G. Neil , Hansen William , Walter Peter
Published: May 2013

We have used protein-fusions to study in Saccharomyces cerevisiae the topology and integration of arginine permease. Since this membrane protein does not contain a cleavable signal sequence, we sought to identify the first internal signal by, initially, fusing the cytoplasmic enzyme, galactokinase, to various positions along the amino-terminal region, and then measuring in vitro the translocation of galactokinase across the membrane of the endoplasmic reticulum. Five fusion proteins were examined that contained, progressively, zero to four hydrophobic segments. The galactokinase moiety of fusion 5, but not fusions 1—4, was translocated. Fusion 4 differed from 5 by only the fourth hydrophobic segment, indicated that this region contains the first internal signal. From this we conclude that hydrophobic segment IV spans the membrane, and that the hydrophilic domain amino-terminal to it lies on the cytoplasmic side of the membrane. If any of the first three segments actually span the membrane, then they probably integrate by a mechanism that differs from segment IV.
Gene products required for chromosome separation
Authors: Yanagida Mitsuhiro
Published: May 2013

Gene products required for mitotic chromosome separation in the .fission yeast Schizosaccharo- myces pombe are described. They have been identified by two distinct strategies of mutant isolation, followed by gene cloning and immunochemical characterization of gene products. The roles of four representative genes, namely nda3+, nuc2+, top2+ and dis2+, encoding β-tubulin, a nuclear scaffold-like protein, DNA topoisomerase II and type-1 protein phosphatase, respectively, are discussed in regard to the mechanisms and control of chromosome separation.
The mammalian cdc2 protein kinase: mechanisms of regulation during the cell cycle
Authors: Draetta Giulio , Beach David
Published: May 2013

Recent experimental evidence has demonstrated the central role of the cdc2 protein kinase in the transition from G2 to M phase in eukaryotic cells. We shall review our knowledge of the mechanisms which coordinate activation of the kinase with cell cycle-specific events in mammalian cells.
The role of cyclin synthesis, modification and destruction in the control of cell division
Authors: Minshull Jeremy , Pines Jon , Golsteyn Roy , Standart Nancy , Mackie Shaun , Colman Alan , Blow Julian , Ruderman Joan V. , Wu Mike , Hunt Tim
Published: May 2013

This paper reviews our current knowledge of the cyclins based on observations of the oocytes and eggs of sea urchins, clams and frogs. Cyclins are proteins found in all eukaryotes whose special property is rapid destruction at specific stages in the cell cycle. The cyclins fall into three families. A-type cyclins have been found in clams, flies and frogs. B-type cyclins have been found in clams, flies, frogs, sea urchins and fission yeast. A more distantly related family of three genes is found in Saccharomyces cerevisiae. B-type cyclins appear to be required for cells to enter mitosis, and their destruction is thought to be necessary for exit from mitosis. We describe evidence in support of these ideas, and describe various conditions under which cyclin destruction is delayed or deranged. We conclude with a discussion of the relationship between the cyclins and maturation- (or M phase-) promoting factor and some ideas on how the cyclins may work.
Nuclear envelope dynamics and nucleocytoplasmic transport
Authors: Stewart Murray , Whytock Sue , Moir Robert D.
Published: May 2013

We have combined structural, biochemical and recombinant DNA methods to explore molecular interactions involved in nuclear envelope assembly dynamics and nucleocytoplasmic transport. Electron microscopy has established the overall architecture of the envelope and the relationship between nuclear pores, lamina fibres and pore-connecting fibrils. The lamin proteins that constitute the lamina resemble intermediate filament proteins, and assemble and disassemble during mitosis in response to phosphorylation. Lamins have been expressed in E. coli to facilitate structural investigations and the exploration of interaction sites with other envelope components. Disruption of envelopes has shown that nuclear pores are constructed from a central cylinder with cytoplasmic and nucleoplasmic rings. Examination of envelopes transporting gold-labelled nucleoplasmin has indicated that the transport pathway is complex and probably involves ring components in addition to the central cylinder. Molecular motors may be involved in changes in pore shape to enable transport and in the translocation mechanism.
Molecular biology of actin binding proteins: evidence for a common structural domain in the F-actin binding sites of gelsolin and α-actinin
Authors: Way Michael , Pope Brian , Weeds Alan
Published: May 2013

We review the impact of molecular biology on actin binding proteins, in particular on sequence relationships and expression of clones to dissect properties in vitro. Significant homologies exist between proteins in each class, but we propose, in addition, that common structural features exist between the F-actin binding sites of severing and cross-linking proteins.
Aluminum fluoride, microtubule stability, and kinesin rigor
Authors: Song Young-Hwa , Heins Susanne , Mandelkow Eckhard , Mandelkow Eva-Maria
Published: May 2013

Aluminum fluoride may be used both to stabilize microtubules and to induce strong binding of kinesin, thus circumventing the need for taxol and AMP-PNP in kinesin preparations.
Small angle X-ray scattering studies on myosin
Authors: Faruqi A. R. , Cross R. A. , Kendrick-Jones J.
Published: May 2013

Small angle X-ray scattering (SAXS) is a potentially powerful method for obtaining structural information from biological molecules in solution. The use of this technique in the laboratory has hitherto been limited by the long exposures necessary to obtain patterns on photographic film. Multi-wire area detectors, due to their high efficiency and absence of noise, enable patterns to be collected much more rapidly, typically in 1-2 h for a typical protein using laboratory sources. This opens up the possibility of using the technique on a semi-routine basis for a wide variety of problems. We outline the use of SAXS to characterise a large conformational change of myosin.
Molecular cloning of protozoan myosin I heavy chain genes
Authors: Hammer John A. , Jung Goeh
Published: May 2013

Myosins I are ubiquitous, nonfilamentous, actin-based mechanoenzymes originally discovered in protozoa. The extensive in vitro biochemical studies of purified protozoan myosins I are now being complemented with in vivo studies using cloned myosin I heavy chain genes and gene targeting techniques. Here we review briefly the systems and methods being used in these efforts to dissect protozoan myosin I structure and function using molecular genetic approaches.
Mechanisms governing neuronal degeneration and axonal regeneration in the mature retinofugal system
Authors: Thanos Solon , Thiel Hans-Jürgen
Published: May 2013

The ability of mammalian CNS neurons to regrow their lesioned axons declines during late embryonic and postnatal development. Consequently, adult retinal ganglion cells of mammals respond to injuries with rapid anterograde and protracted retrograde (Wallerian) degeneration. To monitor the cascade of events initiated by neuronal injuries, and to explore whether the regressive events of this cascade can be blocked or reversed, axotomy-induced ganglion cell responses were investigated in adult rats. The aim of the experiments was to block degradation of axotom-ized ganglion cells with enzymes which inhibit proteolytic activities within the retina (protease inhibitors). To achieve this goal, a new fluorescence technique was employed to assess both the chrono-topological pattern of degradation and the efficacy of the protease inhibitors and anti-inflammatory treatment in preventing cell death. Injection of protease inhibitors alone or combined with dexamethasone into the vitreous body of animals whose optic nerves were transected, protected ganglion cells from degradation and prevented endocytosis-dependent tracing of microglia. Two major functions of rescued ganglion cells proved their viability: (1) the numbers of ganglion cell axons extended from retinal stripes that were explanted 1 week after axotomy and cultured in vitro, were significantly higher when the retinal pieces originated from retinae pretreated with protease inhibitors and dexamethasone at the time of optic nerve transection than from untreated retinae; (2) the numbers of ganglion cells which regenerated axons into transplanted peripheral nerve pieces were more than doubled when the eyes were injected with protease inhibitors and dexamethasone during axotomy. The results show that blocking of the retinal proteases, which are presumably localized in microglial cells, and simultaneous treatment of the intraretinal inflammation, are key steps in understanding the intraretinal responses to axotomy and for beneficially manipulating the numbers of surviving neurons. In addition to the supporting influence of neurotrophic factors and to non-permissive features of oligodendroglia, the microglia co-regulate whether neurons can regenerate their axons.
How to build a glycinergic postsynaptic membrane
Authors: Betz Heinrich , Kuhse Jochen , Schmieden Volker , Malosio Maria-Luisa , Langosch Dieter , Prior Peter , Schmitt Bertram , Kirsch Joachim
Published: May 2013

The inhibitory glycine receptor (GlyR) is a ligandgated chloride channel protein found at many synapses of the mammalian central nervous system. During development, distinct isoforms of the GlyR are generated by the sequential expression of different a subunit variants. The appearance of adult-type GlyRs in spinal cord is accompanied by the accumulation of a 93×103Mr receptor-associated peripheral membrane protein. The latter has been localized at the cytoplasmic face of glycinergic postsynaptic membranes and is thought to anchor GlyRs beneath glycinergic nerve terminals. The 93×103Mr protein binds with high affinity to polymerized tubulin, suggesting that it functions as a receptor-microtubule linking component. Our data suggest that the interaction of developmentally regulated receptor isoforms with specialized microtubule-associated proteins represents a crucial step in the assembly of postsynaptic receptor matrices.
Cyclin A recruits p33cdk2to the cellular transcription factor DRTF1
Authors: Bandara Lasantha R. , Adamczewski Jorg P. , Zamanian Maryam , Poon Randy Y. C. , Hunt Tim , Thangue Nicholas B. La
Published: May 2013

Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdk2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdk2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone Hl kinase activity. Cyclin A cannot, however, direct p34cdk2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.
cis and trans regulation of tissue-specific transcription
Authors: Engel J. D. , Beug H. , Lavail J. H. , Zenke M. W. , Mayo K. , Leonard M. W. , Foley K. P. , Yang Z. , Kornhauser J. M. , Ko L. J. , Lim K.-C. , George K. M. , Breigél K.
Published: May 2013

Analysis of both the cis-regulatory sequences which control globin gene switching as well as the trans-acting factors which bind to these sequences to elicit a differential, developmentally regulated response has lent insight into the general mechanisms responsible for tissue-specific gene regulation. We show here that the chicken adult β-globin gene promoter sequences are intimately involved in competitive interaction with the β/ε-globin enhancer to regulate differentially ε-versus β-globin gene transcription. Secondly, we show that the family of GATA transcription factors directs gene regulation in a variety of discrete cell types, and describe potential cellular target genes for each member of the GATA factor family, as well as potential mechanisms whereby multiple GATA factors expressed in a single cell might be used to elicit differential transcriptional activities.
Transcriptional control by Drosophila gap genes
Authors: Jäckle Herbert , Hoch Michael , Pankratz Michael J. , Gerwin Nicole , Sauer Frank , BröNner GÜNter
Published: May 2013

The segmented body pattern along the longitudinal axis of the Drosophila embryo is established by a cascade of specific transcription factor activities. This cascade is initiated by maternal gene products that are localized at the polar regions of the egg. The initial long-range positional information of the maternal factors, which are transcription factors (or are factors which activate or localize transcription factors), is transferred through the activity of the zygotic segmentation genes. The gap genes act at the top of this regulatory hierarchy. Expression of the gap genes occurs in discrete domains along the longitudinal axis of the preblastoderm and defines specific, overlapping sets of segment primordia. Their protein products, which are DNA-binding transcription factors mostly of the zinc finger type, form broad and overlapping concentration gradients which are controlled by maternal factors and by mutual inter-actions between the gap genes themselves. Once established, these overlapping gap protein gradients provide spatial cues which generate the repeated pattern of the subordinate pair-rule gene expression, thereby blue-printing the pattern of segmental units in the blastoderm embryo. Our results show different strategies by which maternal gene products, in combination with various gap gene proteins, provide position-dependent sets of transcriptional activator/repressor systems which regulate the spatial pattern of specific gap gene expression. Region-specific combinations of different transcription factors that derive from localized gap gene expression eventually generate the periodic pattern of pair-rule gene expression by the direct interaction with individual cis-acting “stripe elements” of particular pair-rule gene promotors. Thus, the developmental fate of blastoderm cells is programmed according to their position within the anterior-posterior axis of the embryo: maternal transcription factors regulate the region-specific expression of first zygotic transcription factors which, by their specific and unique combinations, control subordinate zygotic transcription factors, thereby subdividing the embryo into increasingly smaller units later seen in the larva.
Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain
Authors: Davis Jonathan Q. , Bennett Vann
Published: May 2013

A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypeptides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrins. Major differences in developmental expression have been reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including LI, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrin-binding activity is shared by all of these proteins.