Study of actin filament ends in the human red cell membrane

There is conflicting evidence concerning the state of the actin protofilaments in the membrane cytoskeleton of the human red cell. To resolve this uncertainty, we have analysed their characteristics with respect to nucleation of G-actin polymerization. The effects of cytochalasin E on the rate of elongation of the protofilaments have been measured in a medium containing 0.1 M-sodium chloride and 5 mM-magnesium chloride, using pyrene-labelled G-actin. At an initial monomer concentration far above the critical concentration for the negative ("pointed") end of F-actin, high concentrations of cytochalasin reduce the elongation rate of free F-actin by about 70%. The residual rate is presumed to correspond to the elongation rate at the negative ends. By contrast, the elongation rate on red cell ghosts or cytoskeletons falls to zero, allowing for the background of self-nucleated polymerization of the G-actin. The critical concentration of the actin in the red cell membrane has been measured after elongation of the filaments by added pyrenyl-G-actin in the same solvent. It was found to be 0.07 microM, compared with 0.11 microM under the same conditions for actin alone. This is consistent with prediction for the case of blocked negative ends on the red cell actin. The rate of elongation of actin filaments, free and in the red cell membrane cytoskeleton, has been measured as a function of the concentration of an added actin-capping protein, plasma gelsolin, with a high affinity for the positive ends. The elongation rate falls linearly with increasing gelsolin concentration until it approaches a minimum when the gelsolin has bound to all positive filament ends. The elongation rate at this point corresponds to the activity of the negative ends, and its ratio to the unperturbed polymerization rate (in the absence of capping proteins) is indistinguishable from zero in the case of ghosts, but about 1 : 4 in the case of F-actin. When ATP is replaced in the system by ADP, so that the critical concentrations at the two filament ends are equalized, the difference is equally well-marked: for F-actin, the rate at the equivalence point is about 40% of that in the absence of capping protein, whereas for ghosts the nucleated polymerization rate at the equivalence point is again zero, indicating that under these conditions the negative ends contribute little or not at all to the rate of elongation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The mechanism of cell division

The recently developed approximation method for treating problems of cell biophysics is generalized and corrected in some points. The equations of elongation of a dividing cell of any shape are given for the most general case of finite permeability as well as of finite internal and external diffusion coefficients.     

Engineering synthetic recursive pathways to generate non-natural small molecules

Recursive pathways are broadly defined as those that catalyze a series of reactions such that the key, bond-forming functional group of the substrate is always regenerated in each cycle, allowing for a new cycle of reactions to begin. Recursive carbon-chain elongation pathways in nature produce fatty acids, polyketides, isoprenoids and α-keto acids (αKAs), which all use modular or iterative approaches for chain elongation. Recently, an artificial pathway for αKA elongation has been built that uses an engineered isopropylmalate synthase to recursively condense acetyl-CoA with αKAs. This synthetic approach expands the possibilities for recursive pathways beyond the modular or iterative synthesis of natural products and serves as a case study for understanding the challenges of building recursive pathways from nonrecursive enzymes. There exists the potential to design synthetic recursive pathways far beyond what nature has evolved.     

Dependence of Lettuce Seed Germination on Actinomycin D-resistant RNA Synthesis

The response of Grand Rapids lettuce half seed and intact seed to different nucleic acid and protein synthesis inhibitors was studied. 6-azauracil, 2-thiouracil and cycloheximide were strong inhibitors of germination of intact seed as well as of radicle elongation of half seed. Inhibition by 6-azauracil and 2-thiouracil was reversed by uracil but not by orotic acid, thymine or deoxycytidine. Actinomycin D and puromycin were ineffective suggesting that actinomycin D-resistant RNA synthesis possibly controls both germination of intact seed and radicle elongation of half seed. The requirements of RNA synthesis for the elongation of radicle in half seed in light and in the presence of gibberellic acid was the same. Such was not the case in the germination of intact seed which had different requirements of RNA synthesis in light and in presence of gibberellic acid. It is concluded that the mechanism of gibberellic acid and light in the intact seed germination, in so far as it involves the regulation of RNA synthesis, depends on the integrity of the seed coats.     

The relative significance for stem elongation and flowering in Lolium temulentum of 3β-hydroxylation of gibberellins

In previous experiments with many gibberellins (GAs) and GA derivatives applied to Lolium temulentum L., quite different structural requirements were evident for stem elongation on the one hand and for the promotion of flowering on the other. Whereas hydroxylation at carbons 12, 13 and 15 enhanced flowering relative to stem growth, the reverse was the case at carbon 3 (L.T. Evans et al. 1990, Planta 182, 97–106). The significance of hydroxylation at carbon 3 is examined in this paper. The application of inhibitors of 3β-hydroxylation, including C/D-ring-rearranged GAs, reduced stem growth but, in the case of the two acylcyclohexanediones, increased the flowering response when applied on the inductive long day. Later applications of the acylcyclohexanediones, made after floral initiation had occurred, were inhibitory to flowering, suggesting that subsequent inflorescence development requires 3β-hydroxylated GAs. Applications of the 3α-hydroxy epimers of GA₁, GA₃ and GA₄ gave slightly less promotion of flowering in comparison with the 3β-hydroxy GAs, but far less promotion of stem elongation, except in the case of 3-epi-GA₄, which was comparable to GA₄. The 3α-hydroxy epimer of 2,2-dimethyl GA₄ gave less promotion of flowering than its 3β-hydroxy epimer but almost no promotion of stem elongation. The 3α-hydroxy epimers of GA₃ and 2,2-dimethyl GA₄ did not act as competitive inhibitors of the stem elongation elicited by GA₃ and 2,2-dimethyl GA₄, respectively. These results extend the differences in GA structure which favour flowering as opposed to stem elongation, and indicate that 3-hydroxylation and its epimeric configuration are of much greater importance to stem elongation than to flower initiation in Lolium.     

Electronic energy band structure of uniaxially deformed (5,5) armchair carbon nanotube

Semiempirical molecular orbital calculations of the (5,5) armchair carbon nanotube give the Kekule structure in its ground state with two essentially different bonds (the bond lengths difference is 0.003 nm). This is a result of the Peierls distortions leading to tripled (compared with undistorted case) translational period. When the armchair nanotube is elongated, two first order deformational structural phase transitions are predicted. The first one at the elongation of 5% leads to doubling of a translational period (instead of tripling at smaller elongations). The second one at the elongation of 13% leads to the quinoid type structure. The dependence of the electronic energy-band structure of the (5,5) carbon nanotube on elongation is investigated using the tight binding approximation. The transition from narrow-gap semiconductor to metal is predicted at the elongation of 5%, indicating that the uniaxially deformed armchair carbon nanotube at greater elongation (more than 5%) remains metallic at all temperatures.     

Elastic properties of bacterial flagellar filaments: I. Free rotation case

Elongation of a helical bacterial flagellar filament in a fluid flow with one end attached to a slide glass is calculated. The flagellar filament is regarded as a coil spring. In this case, the spring constant is a function of the elastic constants of the flagellar filament. Relations between the elongation and the elastic constants are discussed.     

Co-ordination between elongation and division in Escherichia coli mediated by the wee gene product

A procedure to construct strains of Escherichia coli containing conditional lethal mutations in two different genes was used to construct a ftsA-3(ts) wee(Am) supF(ts) strain. This strain, OV-25-7, was used to ascertain whether the wee gene product (Wee) acts at the level of regulation of cell elongation or at the co-ordination of elongation and division. The mass per unit length and the buoyant density of cells in the absence of Wee increased only if division was allowed, as in the case of strain OV-25 (wee(Am) supF(ts)), but not when it was inhibited, as in strain OV-25-7. These results suggested that in E. coli the wee gene product was acting at the level of coordination between elongation and cell division.     

The role of secondary mesenchyme cells during sea urchin gastrulation studied by laser ablation

It has long been thought that traction exerted by filopodia of secondary mesenchyme cells (SMCs) is a sufficient mechanism to account for elongation of the archenteron during sea urchin gastrulation. The filopodial traction hypothesis has been directly tested here by laser ablation of SMCs in gastrulae of the sea urchin, Lytechinus pictus. When SMCs are ablated at the onset of secondary invagination, the archenteron doubles in length at the normal rate of elongation, but advance of the tip of the archenteron stops at the 2/3 gastrula stage. In contrast, when all SMCs are ablated at or following the 2/3 gastrula stage, further elongation does not occur. However, if a few SMCs are allowed to remain in 2/3-3/4 gastrulae, elongation continues, although more slowly than in controls. The final length of archenterons in embryos ablated at the 1/3-1/2 gastrula stage is virtually identical to the final length of everted archenterons in LiCl-induced exogastrulae; since filopodial traction is not exerted in either case, an alternate, common mechanism of elongation probably operates in both cases. These results suggest that archenteron elongation involves two processes: (1) active, filopodia-independent elongation, which depends on active cell rearrangement and (2) filopodia-dependent elongation, which depends on mechanical tension exerted by the filopodia.     

Änderungen im Gehalt an löslichen Zuckern und Zellwandkohlenhydraten während der Hemmung endogenen Zellstreckungswachstums durch Antimetaboliten

Summary The effect of actinomycin D, hydroxyproline, cycloheximide, and chloramphenicol on the soluble sugar and cell-wall carbohydrate content was studied in an effort to look for the primary action of these antimetabolites on the cell-wall in connection with cell elongation during inhibition of endogenous hypocotyl growth in mustard seedlings (Sinapis alba L.). The experiments have been done under steady state conditions as far as the parameters under examination are concerned. During the experimental period hypocotyl elongation is due almost exclusively to cell elongation (Geiser, 1964). Antimetabolite concentrations in an 1 hr feeding period have been chosen to effect about 70% relative inhibition 12 hrs after feeding.All antimetabolites confermably caused hypocotyl inhibition already about 1 hr after the beginning of their application. Cycloheximide and chloramphenicol inhibited or impaired fructose and glucose accumulation 1–2 hrs, and cell-wall carbohydrate synthesis about 3 hrs after the onset of hypocotyl inhibition. In contrast, actinomycin D and hydroxyproline leave fructose and glucose accumulation unchanged up to 9 hrs, but they do inhibit cell-wall carbohydrate synthesis approximately as fast as they inhibit hypocotyl elongation. However, the relative inhibition of cell-wall carbohydrate synthesis is only 1/3 of the relative inhibition of hypocotyl elongation.A comparison of the lag-phases and the courses of the kinetics reveals that the changes in the soluble sugar and cell-wall carbohydrate content starting 3–4 hrs after antimetabolite application are only secondary changes not directly concerned with the primary processes leading to hypocotyl inhibition. From the far reaching independence of hypocotyl inhibition and cell-wall carbohydrate synthesis during the first hours after feeding, the conclusion can be drawn that in the case of cycloheximide and chloramphenicol the primary inhibition of hypocotyl elongation must be due to changes in the structural arrangement of cell-wall elements and not to any kind of inhibition of the synthesis of cell-wall carbohydrates. In the case of actinomycin D and hydroxyproline also at least the greatest part of the inhibition, if not all of it, must also be mediated by the same process. Though secondary changes observed in soluble sugar and cell-wall carbohydrate content point to rather different patterns of antimetabolite action, the primary action on the cell-wall in connection with cell growth inhibition, according to the present data, seems to be generally the same regardless of which inhibitor is used.     

Thermoelasticity of swollen elastin networks at constant composition

Thermoelastic (force–temperature) measurements were carried out on elastin networks, swollen with a nonvolatile diluent, in elongation over the temperature range 8–35°C. The experiments were conducted at constant composition (thermodynamically closed system) rather than at swelling equilibrium (open system), thus avoiding the need for approximate corrections to account for the changes in swelling with temperature and with elongation. The results indicate that the elastic force is primarily of entropic origin and thus support the random-network model rather than the liquid-drop model for elastin in the rubberlike state. A significant energetic contribution to the force was observed, however, as is usually the case for a variety of elastomeric polymers.     

Elongation as a factor in artefacts of humans and other animals: an Acheulean example in comparative context

Elongation is a commonly found feature in artefacts made and used by humans and other animals and can be analysed in comparative study. Whether made for use in hand or beak, the artefacts have some common properties of length, breadth, thickness and balance point, and elongation can be studied as a factor relating to construction or use of a long axis. In human artefacts, elongation can be traced through the archaeological record, for example in stone blades of the Upper Palaeolithic (traditionally regarded as more sophisticated than earlier artefacts), and in earlier blades of the Middle Palaeolithic. It is now recognized that elongation extends to earlier Palaeolithic artefacts, being found in the repertoire of both Neanderthals and more archaic humans. Artefacts used by non-human animals, including chimpanzees, capuchin monkeys and New Caledonian crows show selection for diameter and length, and consistent interventions of modification. Both chimpanzees and capuchins trim side branches from stems, and appropriate lengths of stave are selected or cut. In human artefacts, occasional organic finds show elongation back to about 0.5 million years. A record of elongation achieved in stone tools survives to at least 1.75 Ma (million years ago) in the Acheulean tradition. Throughout this tradition, some Acheulean handaxes are highly elongated, usually found with others that are less elongated. Finds from the million-year-old site of Kilombe and Kenya are given as an example. These findings argue that the elongation need not be integral to a design, but that artefacts may be the outcome of adjustments to individual variables. Such individual adjustments are seen in animal artefacts. In the case of a handaxe, the maker must balance the adjustments to achieve a satisfactory outcome in the artefact as a whole. It is argued that the need to make decisions about individual variables within multivariate objects provides an essential continuity across artefacts made by different species.     

Assembly of tobacco mosaic virus in vitro. Improved model for the elongation process by protein subunits.

The in vitro assembly reaction of tobacco mosaic virus (TMV), especially the elongation process of partially reconstituted RNA (PRR) by protein subunits, was observed by electron microscopy. After addition of TMV-protein subunits, the PRR appeared as rods with a clump at one end, believed to be a complex between added protein subunits and the RNA tail protruding from PRR. The subunits entrapped on the RNA tails in the forms of clumps were progressively incorporated into the growing rods on incubation, ending with the formation of completely reconstituted rods. The clumps were also observed after addition of cucumber green mottle mosaic virus (CGMMV) protein subunits to rods partially reconstituted from RNA and TMV-protein. In this case, the protein subunits, seen as clumps, did not become incorporated to form elongating rods. An improved model for the elongation of TMV rods is proposed. The elongation process is composed of two steps, with the first step being the interaction of protein subunits with the RNA tail protruding from the growing rod. Any protein having a specific binding site for TMV-rna, not limited to TMV-protein, will react in the first step. The second step is the incorporation of the protein on the RNA tail into a rod-shaped structure, with consequent elongation of the growing rod. It appears that only protein homologous with that in the partially reconstituted rods can partake in the second step.     

What determines eukaryotic translation elongation: recent molecular and quantitative analyses of protein synthesis

Protein synthesis from mRNA is an energy-intensive and tightly controlled cellular process. Translation elongation is a well-coordinated, multifactorial step in translation that undergoes dynamic regulation owing to cellular state and environmental determinants. Recent studies involving genome-wide approaches have uncovered some crucial aspects of translation elongation including the mRNA itself and the nascent polypeptide chain. Additionally, these studies have fuelled quantitative and mathematical modelling of translation elongation. In this review, we provide a comprehensive overview of the key determinants of translation elongation. We discuss consequences of ribosome stalling or collision, and how the cells regulate translation in case of such events. Next, we review theoretical approaches and widely used mathematical models that have become an essential ingredient to interpret complex molecular datasets and study translation dynamics quantitatively. Finally, we review recent advances in live-cell reporter and related analysis techniques, to monitor the translation dynamics of single cells and single-mRNA molecules in real time.     

On the mechanics of viscous bodies and elongation in ellipsoidal cells

After deriving some auxiliary equations for the average elongation of a viscous body under the action of forces derived from a potential, the diffusion problem for an ellipsoidal cell with a constant rate of reaction is solved for the case of an infinite permeability. The equation of elongation of such a cell under the influence of diffusion forces is derived, and compared with the, approximate expression obtained by N. Rashevsky for any kind of oblong cell. The two equations are in fair agreement. Effects of constant and variable surface tension are studied.     

Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3' end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq-RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).     

The unfolding story of polypeptide release factors

Two recent cryo-EM reconstructions of the ribosome-bound release factor RF2 reveal an open, tri-lobed shape of RF2, in contrast to the comma-shaped molecule seen in the crystal structure. This indicates that RF2 undergoes a conformational change upon binding to the ribosome. Moreover, RF2 does not seem to be a molecular mimic of tRNA as is the case for elongation factor G.     

The role actin filaments play in providing the characteristic curved form of Drosophila bristles

Drosophila bristles display a precise orientation and curvature. An asymmetric extension of the socket cell overlies the newly emerging bristle rudiment to provide direction for bristle elongation, a process thought to be orchestrated by the nerve dendrite lying between these cells. Scanning electron microscopic analysis of individual bristles showed that curvature is planar and far greater near the bristle base. Correlated with this, as development proceeds the pupa gradually recedes from the inner pupal case (an extracellular layer that encloses the pupa) leading to less bristle curvature along the shaft. We propose that the inner pupal case induces elongating bristles to bend when they contact this barrier. During elongation the actin cytoskeleton locks in this curvature by grafting together the overlapping modules that comprise the long filament bundles. Because the bristle is curved, the actin bundles on the superior side must be longer than those on the inferior side. This is accomplished during grafting by greater elongation of superior side modules. Poor actin cross-bridging in mutant bristles results in altered curvature. Thus, the pattern of bristle curvature is a product of both extrinsic factors-the socket cell and the inner pupal case--and intrinsic factors--actin cytoskeleton assembly.