Lysosomal uptake of isolated cell organelles microinjected into HeLa cells

Lysosomes and microsomes were isolated from rat liver and microinjected into the cytoplasm of HeLa cells. The fate of the transplanted organelles and their effects on the recipient cells were followed in the electron microscope at various time intervals after administration. Needle injection with buffer or sucrose did not seem to evoke any ultrastructural alterations, such as induced autophagy or other signs of sublethal cell injury. Recipients of microinjected cell organelles elicited a rapid and conspicuous increase in membrane-bounded cytoplasmic vacuoles, concomitant with the disappearance of the injected material. Golgi complexes became abundant with many small vesicles clustering around their cisternae. The volume density of the lysosomal compartment increased 2-3-fold after organelle injection as compared with control-injected (0.3 M sucrose) or noninjected cells. Our preliminary results show that isolated cell organelles can be microinjected into cells n culture and indicate that the microinjected organelles were segregated from the cytoplasm into membrane-bounded vacuoles probably through autophagolysosome formation. Thus, this technique offers an additional approach for studies on the segregation and degradation of cell organelles in somatic cells and may enable more detailed analyses on the mechanisms of autophagic sequestration of specific cell organelles.     

Evolutionary origin of cell organelles]

A review on the evolutionary origin of the energy-yielding eukaryotic organelles is presented. Current autogenetic (endogenous compartmentalization) schemes, as well as different variants of symbiogenesis, are critically envisaged. A new symbiogenetic scheme is put forth, according to which mitochondria and chloroplasts originated divergently from a primordial photosynthetic organelle; the latter was acquired by endosymbiosis of ancient cyanobacteria in the cells of protoeukaryotes.     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

Seed plant phylogeny and the origin of angiosperms: An experimental cladistic approach

We present a numerical cladistic (parsimony) analysis of seed plants plus progymnosperms, using characters from all parts of the plant body, outgroup comparison, and a method of character coding that avoids biases for or against alternative morphological theories. The robustness of the results was tested by construction of alternative trees and analysis of subsets of the data. These experiments show that although some clades are strongly supported, they can often be related to each other in very different but nearly equally parsimonious ways, apparently because of extensive homoplasy. Our results support Rothwell’s idea that coniferopsids are derived fromCallistophyton- like platyspermic seed ferns with saccate pollen, but the hypothesis that they evolved fromArchaeopteris- like progymnosperms and the seed arose twice is nearly as parsimonious. Meyen’s division of seed plants into radiospermic and primarily and secondarily platyspermic lines is highly unparsimonious, but his suggestion that ginkgos are related to peltasperms deserves attention. Angiosperms belong among the platyspermic groups, as the sister group of Bennettitales,Pentoxylon, and Gnetales, and this “anthophyte” clade is best related toCaytonia and glossopterids, although relationships with other combinations of Mesozoic seed fern taxa are nearly as parsimonious. These results imply that the angiosperm carpel can be interpreted as a modified pinnate sporophyll bearing anatropous cupules (=bitegmic ovules), while gnetalian strobili are best interpreted as aggregations of highly reduced bennettitalian flowers, as anticipated by Arber and Parkin and Crane. Our most parsimonious trees imply that the angiosperm line (though not necessarily all its modern features) extended back to the Triassic, but a later derivation of angiosperms from some species ofCaytonia or Bennettitales, which would be nearly as parsimonious, should also be considered. These results raise the possibility that many features considered key adaptations in the origin and rise of angiosperms (insectpollinated flowers, rapid reproduction, drought tolerance) were actually inherited from their gymnospermous precursors. The explosive diversification of angiosperms may instead have been a consequence of carpel closure, resulting in increased speciation rates due to potential for stigmatic isolating mechanisms and/or new means of dispersal. DNA sequencing of extant plants and better information on anatomy, chemistry, sporophyll morphology, and embryology of Bennettitales and Caytoniales and the morphological diversity of Mesozoic anthophytes could provide critical tests of relationships.     

An evolutionary approach to the traveling salesman problem

Evolutionary optimization has been proposed as a method to generate machine learning through automated discovery. A simulation of natural evolution is conducted using the traveling salesman problem as an artificial environment. For an exact solution of a traveling salesman problem, the only known algorithms require the number of steps to grow at least exponentially with the number of elements in the problem. Three adaptive techniques are described and analyzed. Evolutionary adaptation is demonstrated to be worthwhile in a variety of contexts. Local stagnation is prevented by allowing for the probabilistic survival of the simulated organisms. In complex problems, the final routing is estimated to be better than 99.99999999999% of all possible tours, even though only a small fraction (8.58 × 10^–151) of the total number of tours are examined.     

An evolutionary scenario for the origin of flowers

The Mostly Male theory is the first to use evidence from gene phylogenies, genetics, modern plant morphology and fossils to explain the evolutionary origin of flowers. It proposes that flower organization derives more from the male structures of ancestral gymnosperms than from female structures. The theory arose from a hypothesis-based study. Such studies are the most likely to generate testable evolutionary scenarios, which should be the ultimate goal of evo-devo.     

An evolutionary approach to enzyme kinetics: Optimization of ordered mechanisms

A theoretical investigation is presented which allows the calculation of rate constants and phenomenological parameters in states of maximal reaction rates for unbranched enzymic reactions. The analysis is based on the assumption that an increase in reaction rates was an important characteristic of the evolution of the kinetic properties of enzymes. The corresponding nonlinear optimization problem is solved taking into account the constraint that the rate constants of the elementary processes do not exceed certain upper limits. One-substrate-one-product reactions with two, three and four steps are treated in detail. Generalizations concern ordered uni-uni-reactions involving an arbitrary number of elementary steps. It could be shown that depending on the substrate and product concentrations different types of solutions can be found which are classified according to the number of rate constants assuming in the optimal state submaximal values. A general rule is derived concerning the number of possible solutions of the given optimization problem. For high values of the equilibrium constant one solution always applies to a very large range of the concentrations of the reactants. This solution is characterized by maximal values of the rate constants of all forward reactions and by non-maximal values of the rate constants of all backward reactions. Optimal kinetic parameters of ordered enzymic mechanisms with two substrates and one product (bi-uni-mechanisms) are calculated for the first time. Depending on the substrate and product concentrations a complete set of solutions is found. In all cases studied the model predicts a matching of the concentrations of the reactants and the corresponding Michaelis constants, which is in good accordance with the experimental data. It is discussed how the model can be applied to the calculation of the optimal kinetic design of real enzymes.     

26 Inversion approach to the origin of life: theoretical notions and experimental data

The essence of the inversion concept of the origin of life can be narrowed down to the following theses: (1) thermodynamic inversion is the key transformation of prebiotic microsystems leading to their transition into primary forms of life; (2) this transformation might occur only in the microsystems oscillating around the bifurcation point under far-from-equilibrium conditions. The transformation consists in the inversion of the balance “free energy contribution entropy contribution” (as well as “information contribution informational entropy contribution”), from negative to positive values. At the inversion moment, the microsystem radically reorganizes in accordance with the new negentropy (i.e. biological) way of organization. According to this concept, the origin-of-life process on the early Earth took place in oscillating hydrothermal medium. The process was taking two successive stages: (1) spontaneous self-assembly of initial three-dimensional prebiotic microsystems composed mainly of hydrocarbons, lipids, and simple amino acids, or their precursors, within the temperature interval of 100–300?°C (prebiotic stage); (2) nonspontaneous synthesis of sugars, ATP, and nucleic acids started at the inversion moment under the temperature 70–100?°C (biotic stage). Macro and microfluctuations of thermodynamic and physicochemical parameters able to sustain this way of chemical conversion have been detected in several contemporary hydrothermal systems (Kompanichenko, 2012). Conditions in potential hydrothermal medium for the origin of life were explored on the examples of several hydrothermal systems in Kamchatka peninsula. Temperature of water in hot springs ranges from?<?60 to 98?°C, in the bore holes water-steam temperature varies from?<?100 to 239?°C, and pressure from?<?1 to 35 bars at the wellheads; pH is within the interval 2.5–9.0. Pressure monitoring at the depth 950?meters in the borehole No. 30 (Mutnovsky field) has revealed high-amplitude (up to 1–2 bars) irregular macrofluctuations and low-amplitude quite regular microoscillations of pressure (amplitudes 0.1–0.3 bars). Hydrocarbons, lipid precursors, and simple amino acids are available in the fluid. The lifeless condensate of water-steam mixture (temperature 108–175?°C) contains aromatic hydrocarbons, n-alkanes, ketons, alcohols, and aldehydes. In addition to those, cycloalkanes, alkenes, dietoxyalkanes, naphtenes, fatty acids, ethyl ethers of fatty acids, and monoglycerides have been detected in hot solutions inhabited by thermophiles and hyperthermophiles (temperature 70–98?°C). According to Mukhin et al. (1979), glycine of probably abiotic origination was detected in lifeless condensate.     

Transitions and transversions in evolutionary descent: An approach to understanding

Summary In this paper I lay a quantitative theoretical groundwork for understanding the proportions of the possible types of base substitutions observed between 12 genes sharing a common ancestor and isolated from extant species. The experimentally observed types of base substitution between two sequenced genes do not give a direct measure of the types of base substitutions that occur during evolutionary descent. However, by use of a statistical assemblage of these observations, we can recover, without the assumption of parsimony, the conditional base substitution probabilities that determine this descent. Three methods—direct count, regression, and informational entropy maximization—are described by which these probabilities can be estimated from experimental data. The methods are complementary in that each is most useful for somewhat different types of experimental data. These methods are used to study the ratio of transversions to transitions during gene divergence. Though this ratio is not constant during divergence, it does approach a stable limiting value that in principle can vary from zero, corresponding to 100% transition differences, to infinity, corresponding to 0% transition differences. In practice the limiting ratio tends to hover around a value of two, which is expected on a random basis. However, base substitution pathways that are very nonrandom also may lead to a limiting ratio of exactly two, so that such a value is not diagnostic for random pathways. The limiting ratio can be directly calculated from a knowledge of the twelve conditional probabilities for each type of base substitution, or from a knowledge of the equilibrium base composition of the DNAs compared. An expression is given for this calculation. Fifteen years ago Jean Derancourt, Andrew Lebor and Emile Zuckerkandl (1967), analyzing the amino acid sequence of globin chains coded by nuclear genes, made the original observation that the proportion of transition differences decreases with increasing evolutionary time. Recently Brown et al. (1982) and Brown and Simpson (1982) have reported a decrease in the observed proportion of transition differences in mitochondrial DNA with increasing evolutionary divergence. The conditions that must be satisfied for this type of behavior to occur at stable base composition and with stable base substitution probabilities are defined. Multiple substitutionsper se do not lead to a decrease in transition differences with increasing evolutionary divergence.     

A phylogenetic approach to gene expression data: evidence for the evolutionary origin of mammalian leukocyte phenotypes

SUMMARY The evolution of multicellular organisms involved the evolution of specialized cell types performing distinct functions; and specialized cell types presumably arose from more generalized ancestral cell types as a result of mutational event, such as gene duplication and changes in gene expression. We used characters based on gene expression data to reconstruct evolutionary relationships among 11 types of lymphocytes by the maximum parsimony method. The resulting phylogenetic tree showed expected patterns including separation of the lymphoid and myeloid lineages; clustering together of granulocyte types; and pairing of phenotypically similar cell types such as T-helper cells type 1 and T-helper cells type 2 (Th1 and Th2). We used phylogenetic analyses of sequence data to determine the time of origin of genes showing significant expression difference between Th1 and Th2 cells. Many such genes, particularly those involved in the regulation of gene expression or activation of proteins, were of ancient origin, having arisen by gene duplication before the most recent common ancestor (MRCA) of tetrapods and teleosts. However, certain other genes with significant expression difference between Th1 and Th2 arose after the tetrapod–teleost MRCA, and some of the latter were specific to eutherian (placental) mammals. This evolutionary pattern is consistent with previous evidence that, while bony fishes possess Th1 and Th2 cells, the latter differ phenotypically in important respects from the corresponding cells of mammals. Our results support a gradualistic model of the evolution of distinctive cellular phenotypes whereby the unique characteristics of a given cell type arise as a result of numerous independent mutational changes over hundreds of millions of years.     

Permeabilized cells as a way of gaining access to intracellular organelles: an approach to glycosylation reactions

The selective plasma membrane permeabilization of animal cells is a way of introducing non permeable substrates into the cytoplasmic space. This technique facilitates the introduction of a wide range of labelled precursors and avoids the drawbacks of subcellular fractionation. We review here various physical and chemical methods successfully used in different metabolic studies, and as an example, note the advantages of permeabilized cells in glycosylation studies.