Question 7: Modelling Minimal ‘Lipid-Peptide’ Cells

This contribution is aimed to give support to ‘bottom-up’ approaches to the minimal or early cell research project. Even if, from this perspective, the most simple living cell still seems very far away, the analysis of less complex, infrabiological cellular systems (some of which could be relatively soon implemented in the lab) probably holds the key, or one of the fundamental keys, to the problem of origins of life. On these lines, we propose a simulation model to study the transition from proto-metabolic ‘lipid’ cells to ‘lipid-peptide’ cells, as a critical step in which self-reproducing vesicles could develop into more functionalized supramolecular systems Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Question 7: Biosynthesis of Phosphatidic Acid in Liposome Compartments – Toward the Self-Reproduction of Minimal Cells

Self-reproduction is one of main properties that define living cells. In order to explore the self-reproduction process for the study of early cells, and to develop a research line somehow connected to the origin of life, we have built up a constructive ‘synthetic cells (minimal cells)’ approach. The minimal cells approach consists in the investigation of the minimal number of elements to accomplish simple cell-like processes – like self-reproduction. Such approach belongs to the field of synthetic biology. The minimal cells are reconstructed from a totally reconstituted cell-free protein synthesis system (PURESYSTEM) and liposome compartments as containers. Based on this approach, we synthesized two membrane proteins (enzymes), GPAT and LPAAT, which are involved in the phosphatidic acid biosynthesis in bacteria. Both membrane proteins were successfully synthesized by PURESYSTEM encapsulated inside POPC liposomes. Additionally, the enzymatic activity of GPAT was restored by mixing the expressed enzyme with lipid and by forming liposomes in situ. Through these experimental evidences, here we present a possible model to achieve self-reproduction in minimal cells. Our results would contribute to the idea that early cells could have been built by an extremely small number of genes. Presented at the International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Neural assemblies and laminar interactions in the cerebral cortex

 Neural assemblies are assumed to become organized and to operate within the cerebral cortex, and so must be constrained by the cytological and physiological properties of this laminated structure. A hypothesis of such assemblies is presented, based on important details of neuronal architecture and physiology in different cortical laminae. Laminae II, III and VI, which are the origin and termination of most cortico-cortical projections, are regarded as the site of storage of most of the information encoded by assemblies – a neuronal ‘library’. Laminae II and III are the most sensitive coincidence detectors, and therefore probably initiate the process of assembly formation. However, these three laminae have very low levels of spontaneous activity in the waking state, and so active cell assemblies cannot base their functioning on these laminae alone. Lamina V pyramidal cells have a much higher level of spontaneous activity. Thus, indirect pathways between ‘library’ cells, via lamina V pyramidal cells, are likely to be more secure than direct ones. It is proposed that direct links between ‘library’ cells become stabilized by Hebbian strengthening, once the recipient ‘library’ cell has been ‘primed’ by neural activity transmitted indirectly via lamina V neurones. Thus lamina V neurones could catalyse the process of assembly formation. Given this proposal, lamina V cells, in their interaction with ‘library’ cells, would code information in terms of precisely timed individual impulses, but would employ a code based on slower frequency changes in their descending influences upon neural centres in the brainstem and spinal cord. Predictions for single unit and electrographic experiments are discussed. Received: 30 November 1995/Accepted in revised form: 3 June 1996     

Genetic control theory of developmental events

An earlier theory of cell differentiation and morphogenesis (Wassermann, 1972, 1973, 1978) is combined with the genetic control model of Davidson and Britten (e.g. 1979). The resulting new theory suggests how, bysystematic process algorithms, specifically enumerated combinations of batteries of structural genes can become switched on in particularly enumerated cells, via battery-specific enumerable regulator genes. The systematization is idealized. Up to a certain stage of development in each mitotically arising cell a unique cell-specific combination of structural genes called ‘marker genes’ is active. Marker genes are assumed to code for cell-specifying marker proteins (CSMPs) which permit cells carrying related markers to recognize each other, thus permitting specific cell sorting.Batteries of marker genes could ensure great developmental precision and can safeguard—via redundancies of CSMP types—against accidental loss or detrimental mutational modification of CSMPs or marker genes, respectively. This paper is much concerned with cell lineage in relation to ‘microdifferentiation’, where ‘microdifferentiation’ of a cell refers to a cell's active marker genes and its syntheses of CSMPs. A drastic distinction is made between ‘microdifferentiation’ and ‘gross’ differentiation of a cell, where the same ‘gross’ differentiation may be shared by a large number of cells that could each be uniquely ‘microdifferentiated’. Typical ‘gross’ differentiation could manifest itself in tissue specificity, whereas, up to certain stages of development, all cells of the same gross differentiation type (say tissue specificity) could each be uniquely ‘microdifferentiated’. The theory also assumes that at certain stages of the developmental process some (or in some organisms all) of the previously uniquely specified cells could give rise to small (or occasionally large) clones of equispecified cells, some of which might form clusters that represent complete ‘morphogenetic fields’ Tentative implementation mechanisms are proposed which suggest how the theory could operate in molecular biological terms. In particular, CSMPs could endow cell surface membranes with a highly specific protein network, and an associated equally specific cell surface coat. It is suggested how these highly specified cell surface coats and other systems could provide an ‘extracellular guidance network’ which could help to direct cells to attain energetically optimal locations relative to each other based on the matching of their surface specificities. In numerous experimental situations, where normally present optimal matching of cells is excluded, ‘alternative matching’ based on experiment-specific suboptimal matching could explain many data, notably in experimental development neurobiology (Wassermann, 1978).     

History of the word photosynthesis and evolution of its definition

In 1893, Charles Barnes (1858–1910) proposed that the biological process for ‘synthesis of complex carbon compounds out of carbonic acid, in the presence of chlorophyll, under the influence of light’ should be designated as either ‘photosyntax’ or ‘photosynthesis.’ He preferred the word ‘photosyntax,’ but ‘photosynthesis’ came into common usage as the term of choice. Later discovery of anoxygenic photosynthetic bacteria and photophosphorylation necessitated redefinition of the term. This essay examines the history of changes in the meaning of photosynthesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

On What It is to Fly Can Tell Us Something About What It is to Live

The plurality of definitions of life is often perceived as an unsatisfying situation stemming from still incomplete knowledge about ‘what it is to live’ as well as from the existence of a variety of methods for reaching a definition. For many, such plurality is to be remedied and the search for a unique and fully satisfactory definition of life pursued. In this contribution on the contrary, it is argued that the existence of such a variety of definitions of life undermines the very feasibility of ever reaching a unique unambiguous definition. It is argued that focusing on the definitions of specific types of ‘living systems’—somehow in the same way that one can define specific types of ‘flying systems’—could be more fruitful from a heuristic point of view than looking for ‘the’ right definition of life, and probably more accurate in terms of carving Nature at its joints.     

A Tribute to Thomas A. Sebeok

According to the approach developed by Thomas A. Sebeok (1921–2001) and his ‘global semiotics,’ semiosis and life converge. This leads to his cardinal axiom: ‘semiosis is the criterial attribute of life.’ His global approach to sign life presupposes his critique of anthropocentrism and glottocentrism. Global semiotics is open to zoosemiotics, indeed, even more broadly, biosemiotics which extends its gaze to semiosis in the whole living universe to include the realms of macro- and microorganisms. In Sebeok’s conception, the sign science is not only the study of communication in culture, but of communicative behaviour from a biosemiotic perspective.     

Triploid banana cell growth phases and the correlation of medium pH changes with somatic embryogenesis in embryogenic cell suspension culture

Embryogenic cell suspensions of Musa AAA and AAB genomic groups were cultured in a maintenance culture medium for 17 generations (lasting for 238 days). The cell growth phases and medium pH changes were also observed correspondingly. Three major growth phases of AAA genomic group have been focused, namely cell releasing, proliferation and globularization phases. During almost all the subculture generations the cell stocks of AAB ‘Raja’ were continuously characterized by proliferating cell aggregates while the globularization phase occurred only for short duration. The medium acidity levels of the cell stocks of AAA ‘Pei-Chiao’ and ‘Robusta’ were commonly scattered in a wider range of pH 3.3–5.3, while the AAB ‘Raja’ were deviated in a narrow range of pH 4.0–4.6. After subculture, culture medium showed biphasic pH changes, which were drastic pH falls followed by an auto-regulated steady-state level. The steady-state pH values in each of the three growth phases (i.e. cell releasing, proliferation and globularization phases) were of 3.3–4.0, 4.0–4.8 and 4.8–5.3 respectively. Morphological bipolarity and the efficiency in the formation of somatic embryos have been thoroughly discussed. Reported research indicates that the condition of pH below 4.6 may prevent the development of embryogenic cells towards polar growth.     

Enrichment of anaerobic nitrate-dependent methanotrophic ‘ Candidatus Methanoperedens nitroreducens’ archaea from an Italian paddy field soil

Paddy fields are a significant source of methane and contribute up to 20% of total methane emissions from wetland ecosystems. These inundated, anoxic soils featuring abundant nitrogen compounds and methane are an ideal niche for nitrate-dependent anaerobic methanotrophs. After 2 years of enrichment with a continuous supply of methane and nitrate as the sole electron donor and acceptor, a stable enrichment dominated by ‘Candidatus Methanoperedens nitroreducens’ archaea and ‘Candidatus Methylomirabilis oxyfera’ NC10 phylum bacteria was achieved. In this community, the methanotrophic archaea supplied the NC10 phylum bacteria with the necessary nitrite through nitrate reduction coupled to methane oxidation. The results of qPCR quantification of 16S ribosomal RNA (rRNA) gene copies, analysis of metagenomic 16S rRNA reads, and fluorescence in situ hybridization (FISH) correlated well and showed that after 2 years, ‘Candidatus Methanoperedens nitroreducens’ had the highest abundance of (2.2 ± 0.4 × 108) 16S rRNA copies per milliliter and constituted approximately 22% of the total microbial community. Phylogenetic analysis showed that the 16S rRNA genes of the dominant microorganisms clustered with previously described ‘Candidatus Methanoperedens nitroreducens ANME2D’ (96% identity) and ‘Candidatus Methylomirabilis oxyfera’ (99% identity) strains. The pooled metagenomic sequences resulted in a high-quality draft genome assembly of ‘Candidatus Methanoperedens nitroreducens Vercelli’ that contained all key functional genes for the reverse methanogenesis pathway and nitrate reduction. The diagnostic mcrA gene was 96% similar to ‘Candidatus Methanoperedens nitroreducens ANME2D’ (WP_048089615.1) at the protein level. The ‘Candidatus Methylomirabilis oxyfera’ draft genome contained the marker genes pmoCAB, mdh, and nirS and putative NO dismutase genes. Whole-reactor anaerobic activity measurements with methane and nitrate revealed an average methane oxidation rate of 0.012 mmol/h/L, with cell-specific methane oxidation rates up to 0.57 fmol/cell/day for ‘Candidatus Methanoperedens nitroreducens’. In summary, this study describes the first enrichment and draft genome of methanotrophic archaea from paddy field soil, where these organisms can contribute significantly to the mitigation of methane emissions.     

Characterization of ‘Schizokinen’; a dihydroxamate-type siderophore produced by Rhizobium leguminosarum IARI 917

The Rhizobia comprise one of the most important groups of beneficial bacteria, which form nodules on the roots (rarely on the stems) of leguminous plants. They live within the nodules and reduce atmospheric nitrogen to ammonia, which is further assimilated by plants into required nitrogenous compounds. The Rhizobia in return obtain nutrition from the plant. Rhizobia are free-living soil bacteria and have to compete with other microorganisms for the limited available iron in the rhizosphere. In order to acquire iron Rhizobia have been shown to express siderophore-mediated iron transport systems. Rhizobium leguminosarum IARI 917 was investigated for its ability to produce siderophore. It was found to produce a dihydroxamate type siderophore under iron restricted conditions. The siderophore was purified and chemically characterized. The ESMS, MS/MS and NMR analysis indicate the dihydroxamate siderophore to be ‘schizokinen’, a siderophore reported to be produced by Bacillus megaterium that shares a similar structure to ‘rhizobactin 1021’ produced by Sinorhizobium meliloti 1021. This is the first report of production of schizokinen by a strain of R. leguminosarum, therefore it was carefully investigated to confirm that it is indeed ‘schizokinen’ and not a degradation product of ‘rhizobactin 1021’. Since ferric–siderophore complexes are transported across the outer membrane (OM) into the periplasm via an OM receptor protein, R. leguminosarum IARI 917 was investigated for the presence of an OM receptor for ‘ferric–schizokinen’. SDS PAGE analysis of whole cell pellet and extracted OM fractions indicate the presence of a possible iron-repressible OM receptor protein with the molecular weight (MW) of approximately 74 kDa.     

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. III. PR-protein gene expression and ethylene evolution in tomato cell lines transgenic for phytohormone-related bacterial genes

 Previous work carried out in our laboratory has shown that, in tomato, the alteration of endogenous phytohormone equilibria through the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis can modify the active defense response to Fusarium oxysporum f. sp. lycopersici. The susceptible cv ‘Red River’ acquires a stable competence for active defense, particularly when the phytohormone equilibrium is altered in favour of cytokinins. Here, we analyse the expression of genes involved in the defense response against pathogens, i.e. pathogenesis-related (PR)-protein genes, in the susceptible ‘Red River’ and resistant ‘Davis’ cultivars transgenic for the aforementioned genes. Fungal cell-wall components, glutathione, salicylic acid and the ethylene-forming ethephon are used as “probes” for the induction of defense processes, including ethylene production. The data obtained show that the extracellular PR-proteins (acidic chitinase and PR-1 protein) that were inducible in the control tissue of the resistant ‘Davis’ cultivar and not expressed in the susceptible ‘Red River’ cultivar became constitutive in the transgenic tissues of both. On the other hand, expression of the intracellular PR-proteins (basic chitinase and β-1,3-glucanase) was found to be constitutive in all cases, both in the control and in the transgenic cell lines of the resistant and the susceptible tomato cultivars. Ethylene production was higher in ‘Davis’ than in ‘Red River’, and significantly increased in the transgenic cell lines, particularly when cytokinin synthesis was altered. Received: 25 February 1998 / Accepted: 7 April 1998     

Photoperiodic control of larval development and wing form inModicogryllus sp. (Orthoptera: Gryllidae)

The bivoltine cricket locally known as ‘Tambo-Koorogi’ (Modicogryllus sp.) hibernates as a half-grown larva, and shows a developmental response to changing as well as stationary photoperiod. At 25°C, larvae matured in about 7–10 weeks in continuous or 16 h daily light, but took 10–20 weeks or more in 14 h and shorter days. A day of 15 h exerted an intermediate effect, giving a development time of 7–17 weeks. However, an increase in daylength from 12 to 15 h in the early larval life prevented diapause, while a decrease from 15 to 12 h retarded development more than a constant 12 h day. Short-winged adults occurred only when the photophase was 15 h or shorter, and their frequency increased with decreased daylength. Although the short-winged form took longer to mature than the long-winged form under the same conditions of photoperiod, delayed growth was not always associated with brachypterism.     

Activities of sucrolytic enzymes in developing pods of lentil genotypes differing in seed size

The activities of sucrolytic enzymes viz. sucrose synthase and invertases were compared in developing pods of two genotypes of lentil differing in seed weight. Biomass accumulation of both the podwall and seed of ‘large’ genotype was higher during development as compared to the ‘small’ genotype. High activity of acid invertase together with prolonged activity of alkaline invertase in podwall of ‘large’ genotype may lead to longer cell division phase resulting in its larger size and biomass. Greater biomass of podwall could be responsible for providing more reserves for the developing seed hence determining its size. Higher alkaline invertase activity in ‘large’ seed from 15–20 DAF can be correlated to the sustained sucrolytic conditions for producing more cells required for its larger size. Increased levels of sucrose synthase in ‘large’ seed especially during maturation phase suggest the role of this enzyme in enhancing the seed sink strength.     

Distribution and dynamics of bacterioplankton production in a polymictic tropical lake (Lago Xolotlán,Nicaragua)

From 1988 to 1993 we assessed the variability of bacterioplankton production and biomass in Lake Xolotlán (L. Managua), Nicaragua via [3H]thymidine incorporation into DNA and cell counting. Bacterial production ranged from 3 to 8 μg C l-1 h-1, and since production was equal throughout the water column, areal production was high (≈ 600–1200 mg C m-2 d-1). Bacterial abundance in Lake Managua was extremely high, 7–30 × 109 cells l-1. Thus, specific rates of bacterial production were low. There was a strong correlation between production and number and the specific rate of bacterial production was constant. Comparable measurements of production via [3H]leucine incorporation into proteins indicated that bacteria were experiencing ‘balanced growth’. We conclude that bacterioplankton in Lake Xolotlán had reached its carrying capacity and a significant correlation between bacterial production and concentration of phaeophytin implied that dead or dying algae was the limiting substrate for bacterioplankton. The majority of bacterial number and most of bacterial production (up to 75%) were associated with particles in the >3-μm fraction, probably lysing algal cells to which bacterioplankton were ‘attached’. Grazing on bacterioplankton must be low and bacteria should be a ‘sink’ for organic matter in Lake Xolotlán. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosomal control of glutenin subunits in aneuploid lines of wheat: analysis by reversed-phase high-performance liquid chromatography

Summary Glutenin subunits from nullisomic-tetrasomic and ditelocentric lines of the hexaploid wheat variety ‘Chinese Spring’ (CS) and from substitution lines of the durum wheat variety ‘Langdon’ were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) at 70 °C using a gradient of acetonitrile in the presence of 0.1% trifluoroacetic acid. Nineteen subunits were detected in CS. The presence and amounts of four early-eluted subunits were found, through aneuploid analysis, to be controlled by the long arms of chromosomes 1D (1DL) (peaks 1–2) and 1B (1BL) (peaks 3–4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that these four subunits are the high molecular weight subunits of glutenin, which elute in the order 1Dy, 1Dx, 1By, and 1Bx. Similar amounts of 1DL subunits were present (6.3 and 8.8% of total glutenin), but 1BL subunits differed more in abundance (5.4 and 9.5%, respectively). Results indicate that most late-eluting CS glutenin subunits were coded by structural genes on the short arms of homoeologous group 1 chromosomes: 6 by 1DS, 5 by 1AS, and 4 by 1BS. Glutenin of tetraploid ‘Langdon’ durum wheat separated into nine major subunits: 6 were coded by genes on 1B chromosomes, and 3 on 1A chromosomes. Gene locations for glutenin subunits in the tetraploid durum varieties ‘Edmore’ and ‘Kharkovskaya-5’ are also given. These results should make RP-HPLC a powerful tool for qualitative and quantitative genetic studies of wheat glutenin. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned Stationed at the Northern Regional Research Center, Peoria.     

Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro

Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of function’ approaches in vitro. This revised version was published online in July 2006 with corrections to the Cover Date.     

Targeting an anti-viral CD8+ T cell response to a growing tumor facilitates its rejection

 We demonstrate in a murine model that targeting an anti-viral T cell response to a growing tumor facilitates priming of a tumor-associated antigen (TAA)-specific, rejecting T cell response. Murine P815 mastocytoma cells grow aggressively in a syngeneic host. Transfected P815/S cells (expressing the hepatitis B surface antigen, HBsAg) also grow as subcutaneous tumors, but occasional ‘spontaneous’ rejections after transient growth are observed. Growth of P815/S tumors (but not of P815 tumors) is efficiently suppressed by a CD8⁺ cytotoxic T lymphocyte (CTL)-dependent immune mechanism in mice primed to HBsAg by DNA–immunization. In hosts immunized against HBsAg by DNA vaccination, HBsAg-specific CTL are generated. This specific CTL reactivity was targeted to s.c.-growing P815 tumors by intra tumor injections of either HBsAg-encoding plasmid DNA or viable P815/S cells; this treatment led to tumor rejection in 70–80% of the tumor-bearing animals. All rejecting animals showed a CD8⁺ CTL-dependent resistance to subsequent challenges by native, non-transfected P815 tumors. Targeting an established anti-viral (‘strong’) CTL response to a growing tumor hence is an efficient strategy to facilitate priming of a rejecting CTL response against (‘weak’) TAA in this system. Received: 18 December 1996 / Accepted: 6 February 1997