Trypsin crystallization by membrane-based techniques

To grow protein crystals is not an easy task; moreover, if we need to grow protein crystals with controlled shape, size, and size distribution, depending on their application, the mission becomes even harder. Membrane crystallization has been recognized as an interesting tool for growing protein crystals with enhanced crystallization kinetics, both in static and in forced solution flow configuration, without detrimental effects on crystal quality. In the present work, we have studied the membrane crystallization process of benzamidine inhibited trypsin from bovine pancreas (BPT), with ammonium sulphate (dissolved in Tris-HCl buffer, 0.1 M, pH 8.5), as precipitant agent. We have demonstrated that, by using the membrane crystallization technique, BPT crystals can be obtained in 24-48 h, in static configuration, and in 4-7 days, in a forced solution flow system, depending on the experimental conditions. Furthermore, the kinetics of BPT crystallization have been modulated, to control the morphological characteristics of the crystals produced, by an accurate selection of the operative parameters involved in the process. The active membrane surface and the flow rate of extraction solvent in quiescent configuration, and the solution velocity in forced convection solution experiments, were the parameters investigated. In this respect, membrane crystallization techniques have been assessed as an interesting way for growing proteins, and more specifically enzyme crystals, with high control on the final properties of the crystalline material produced, with potential fundamental implication in the field of structural biology and biotechnology.     

Type V myosin focuses the polarisome and shapes the tip of yeast cells

The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin filaments and the fusion of secretory vesicles in yeasts and filamentous fungi. Polarisomes are compact, spotlike structures at the growing tips of their respective cells. The molecular forces that control the form and size of this microcompartment are not known. Here we identify a complex between the polarisome subunit Pea2 and the type V Myosin Myo2 that anchors Myo2 at the cortex of yeast cells. We discovered a point mutation in the cargo-binding domain of Myo2 that impairs the interaction with Pea2 and consequently the formation and focused localization of the polarisome. Cells carrying this mutation grow round instead of elongated buds. Further experiments and biophysical modeling suggest that the interactions between polarisome-bound Myo2 motors and dynamic actin filaments spatially focus the polarisome and sustain its compact shape.     

Uncoupling growth from the cell cycle

How does a Drosophila wing grow to the appropriate size and shape? Although a collaboration of cell division with the patterning of cell fates seems obvious, almost nothing is known about how these two processes are coordinated during development. A recent paper¹ finds that blocking cell division uncouples cell growth from the cell division cycle, displaying remarkable flexibility in the ability of the wing primordia to achieve the right proportions with fewer than normal cells. BioEssays 20: 283-286, 1998.© 1998 John Wiley & Sons, Inc.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin affects fluctuating asymmetry of molar shape in mice, and an epistatic interaction of two genes for molar size

Fluctuating asymmetry (FA), random variation between left and right sides in a bilaterally symmetrical character, is a commonly used measure of developmental instability that is expected to increase with increasing environmental stress. One potential stressor is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a powerful toxicant known to disturb tooth development. In this study, mice in the F(2) generation produced from an intercross between two inbred strains (C57BL/6J and AKR/J) were exposed in utero to TCDD. We hypothesized that TCDD would increase FA in the molars of exposed mice over that of the control mice. In addition, we hypothesized that we would discover genes for molar size, shape or asymmetry whose expression would be affected by TCDD. We detected a very small, but significant, increase in FA of molar shape (but not size) in the TCDD-exposed mice compared to the control mice, although molar size and shape did not differ between these groups. Although we did not uncover any genes that acted differently in the TCDD exposed and control groups, we did identify two genes whose dominance by additive epistatic effect on molar size was affected by TCDD. We concluded that although TCDD may be affecting the expression of some genes governing the development of molars in our population of mice, FA of molar size and shape is not a particularly sensitive indicator of this effect.     

Polyhydroxyalkanoate (PHA) granule formation in<Emphasis Type="Italic"> Ralstonia eutropha</Emphasis> cells: a computer simulation

Computer simulation of polyhydroxyalkanoate (PHA) granule formation in vivo could help to design strategies to optimize the fermentation process and achieve higher yields of PHA. It could also suggest biotechnological approaches to control the granule size and molecular weight of the polymer. A computer program simulating the formation of PHA granules inside a Ralstonia eutropha cell was developed, based on published experimental data. The results are applicable to R. eutropha cells or other microorganisms and transgenic plants, where polyhydroxybutyrate production is made possible by heterologous expression systems. The simulation starts at the outset of the PHA accumulation phase when the cells are small and contain no PHA granules. In the presence of abundant glucose, the cell responds to phosphorus limitation by producing 3-hydroxybutyryl-CoA which undergoes polymerization on the few PHA synthase molecules present in the cytoplasm. The amphiphilic PHA synthase–PHA complex attracts additional PHA synthase molecules and granules begin to grow from these initiation sites. Phosphorus limitation and the appearance of PHA in the cytoplasm also stimulate production of phasin molecules that attach themselves to the growing granules. As the granules grow bigger, they begin to touch each other and move to optimize their packing. The phasin coat prevents the granules from coalescing. The size of the cell increases and its prolate ellipsoid shape becomes closer to spherical. The accumulation process stops either when the supply of glucose is exhausted or when the granules become tightly packed within the cell, so that access to their surface is limited. All important variables, such as cell dimensions, granule size, counts of granule-associated molecules, PHA yield, degree of polymerization of the PHA molecules, etc., are recorded in real time during the simulation. Examples of virtual experiments with the cell and their results are shown.     

The island rule: made to be broken?

The island rule is a hypothesis whereby small mammals evolve larger size on islands while large insular mammals dwarf. The rule is believed to emanate from small mammals growing larger to control more resources and enhance metabolic efficiency, while large mammals evolve smaller size to reduce resource requirements and increase reproductive output. We show that there is no evidence for the existence of the island rule when phylogenetic comparative methods are applied to a large, high-quality dataset. Rather, there are just a few clade-specific patterns: carnivores; heteromyid rodents; and artiodactyls typically evolve smaller size on islands whereas murid rodents usually grow larger. The island rule is probably an artefact of comparing distantly related groups showing clade-specific responses to insularity. Instead of a rule, size evolution on islands is likely to be governed by the biotic and abiotic characteristics of different islands, the biology of the species in question and contingency.     

Biomineralization of limpet teeth: a cryo-TEM study of the organic matrix and the onset of mineral deposition

The continuously growing limpet radula contains teeth at various stages of maturity and thus provides an excellent opportunity for studying the processes and mechanisms of their mineralization. We report here on our structural investigations of the pre-formed chitin matrix and the initial deposition and growth of goethite (α-FeOOH) crystals within the matrix. By using cryo-techniques, in which unstained sections of the teeth are examined in a frozen-hydrated state in a transmission electron microscope (TEM), we were able to characterize the process without introducing artifacts normally associated with the staining, dehydration, and embedding required for conventional TEM. The unmineralized matrix consists of relatively well ordered, densely packed arrays of chitin fibers, with only a few nanometers between adjacent fibers. There are clearly no pre-formed compartments that control goethite crystal size and shape; rather, crystals must push aside or engulf the fibers as they grow. By examining teeth nearly row-by-row around the onset of mineralization, we were able to image the first-formed mineral within the chitin matrix. These linear deposits of goethite appear to nucleate on the chitin fibers, which thus control the orientation of the crystals. Crystal growth, on the other hand, is apparently not influenced by the matrix, in contrast to many other biomineralization systems.     

Cell shape and division in Escherichia coli: experiments with shape and division mutants.

Double mutants which carry mutations in genes (rodA, pbpA) required for cell elongation (i.e., maintenance of rod shape) in combination with mutations in genes (ftsA, ftsI, ftsQ, or ftsZ) required for septation were constructed. Such mutants were able to grow for about two mass doublings at a normal rate at the restrictive temperature (42 degrees C). The morphology of the cells formed under these conditions was interpreted by assuming the existence of a generalized system for peptidoglycan growth together with two additional systems which modify the shape of the growing peptidoglycan layer. The results also showed that different fts genes probably control different stages in septation. ftsZ (sulB or sfiB) appears to be required for the earliest step in septation, ftsQ and ftsI (pbpB or sep) are required for a later step or steps, and ftsA is required only for the latest stages in septation.     

De novo growth zone formation from fission yeast spheroplasts

Eukaryotic cells often form polarized growth zones in response to internal or external cues. To understand the establishment of growth zones with specific dimensions we used fission yeast, which grows as a rod-shaped cell of near-constant width from growth zones located at the cell tips. Removing the cell wall creates a round spheroplast with a disorganized cytoskeleton and depolarized growth proteins. As spheroplasts recover, new growth zones form that resemble normal growing cell tips in shape and width, and polarized growth resumes. Regulators of the GTPase Cdc42, which control width in exponentially growing cells, also control spheroplast growth zone width. During recovery the Cdc42 scaffold Scd2 forms a polarized patch in the rounded spheroplast, demonstrating that a growth zone protein can organize independent of cell shape. Rga4, a Cdc42 GTPase activating protein (GAP) that is excluded from cell tips, is initially distributed throughout the spheroplast membrane, but is excluded from the growth zone after a stable patch of Scd2 forms. These results provide evidence that growth zones with normal width and protein localization can form de novo through sequential organization of cellular domains, and that the size of these growth zones is genetically controlled, independent of preexisting cell shape.     

Weather- and population density-induced infantilism in the landsnailTheba pisana in a semi-arid climate

Natural populations of the landsnailTheba pisana (Pulmonata: Helicidae) were studied in the Mediterranean coastal plains of Israel. The life cycle is annual. Egg-laying occurs in the winter and the descendants grow fast during the spring, except for a part of the population the ceases growing. These individuals, termed infantiles, retain immature size and shape and a rudimentary status of the genital system. The percentage of infantilism in the population is positively related to the density of the snail population in the winter, and is negatively related to the humidity of weather in the spring. A natural control mechanism ofT. pisana populations is proposed: (a) in a dense population of young snails infantilism prevents most of them from becoming parents and an over-sized population the following year; (b) in a humid spring a fall in the rate of infantilism enlarges the population size, thus compensating for reduced egg-laying in winter.     

The size of the nucleus increases as yeast cells grow

It is not known how the volume of the cell nucleus is set, nor how the ratio of nuclear volume to cell volume (N/C) is determined. Here, we have measured the size of the nucleus in growing cells of the budding yeast Saccharomyces cerevisiae. Analysis of mutant yeast strains spanning a range of cell sizes revealed that the ratio of average nuclear volume to average cell volume was quite consistent, with nuclear volume being approximately 7% that of cell volume. At the single cell level, nuclear and cell size were strongly correlated in growing wild-type cells, as determined by three different microscopic approaches. Even in G1-phase, nuclear volume grew, although it did not grow quite as fast as overall cell volume. DNA content did not appear to have any immediate, direct influence on nuclear size, in that nuclear size did not increase sharply during S-phase. The maintenance of nuclear size did not require continuous growth or ribosome biogenesis, as starvation and rapamycin treatment had little immediate impact on nuclear size. Blocking the nuclear export of new ribosomal subunits, among other proteins and RNAs, with leptomycin B also had no obvious effect on nuclear size. Nuclear expansion must now be factored into conceptual and mathematical models of budding yeast growth and division. These results raise questions as to the unknown force(s) that expand the nucleus as yeast cells grow.     

Genetic control of epithelial tube size in the Drosophila tracheal system

The proper size of epithelial tubes is critical for the function of the lung, kidney, vascular system and other organs, but the genetic and cellular mechanisms that control epithelial tube size are unknown. We investigated tube size control in the embryonic and larval tracheal (respiratory) system of Drosophila. A morphometric analysis showed that primary tracheal branches have characteristic sizes that undergo programmed changes during development. Branches grow at different rates and their diameters and lengths are regulated independently: tube length increases gradually throughout development, whereas tube diameter increases abruptly at discrete times in development. Cellular analysis and manipulation of tracheal cell number using cell-cycle mutations demonstrated that tube size is not dictated by the specific number or shape of the tracheal cells that constitute it. Rather, tube size appears to be controlled by coordinately regulating the apical (lumenal) surface of tracheal cells. Genetic analysis showed that tube sizes are specified early by branch identity genes, and the subsequent enlargement of branches to their mature sizes and maintenance of the expanded tubes involves a new set of genes described here, which we call tube expansion genes. This work establishes a genetic system for investigating tube size regulation, and provides an outline of the genetic program and cellular events underlying tracheal tube size control.     

Effects of erythrocyte lysate and erythrocyte-conditioned medium on erythroid cells in vitro.

A component of erythrocyte-conditioned medium has been shown to inhibit the incorporation of tritiated thymidine into erythroblasts from fetal mouse liver, proliferating in vitro. This component, however, has no detectable effect on the growth of colonies of erythroid cells stimulated to grow in viscous culture media by the hormone erythropoietin. Erythrocyte lysate and preparations of haemoglobin derived from the lysate increase the number and size of the colonies growing in vitro. Results are discussed in terms of possible control mechanisms in erythropoiesis.     

Heterochrony, maternal effects, and phenotypic variation among sympatric pupfishes

Variation in ontogeny can produce phenotypic variation both within and among species. I investigated whether changes in timing and rate of growth were a source of phenotypic variation in a putative incipient species group of pupfish (Cyprinodon spp.). On San Salvador Island, Bahamas, sympatric forms of pupfish differ in morphology but show only partial reproductive isolation in the laboratory. Offspring from two forms and two geographical areas and their hybrids were bred in the laboratory, and ontogenetic trajectories of their feeding morphology were followed until maturity. In the Bahamian pupfish the two forms grow along similar size but not shape trajectories. Two heterochronic parameters, onset and rate of growth, alter shape trajectories in the Bahamian pupfish. Similar forms from different geographical areas (Florida and the Bahamas) grow along parallel shape trajectories, differing only in one heterochronic parameter, the onset shape. Hybrids within and between the pupfish forms produced intermediate feeding morphologies that were influenced by their maternal phenotype, suggesting that maternal effects may be a source of phenotypic variation in shape that can persist to maturity. In Cyprinodon, small changes in multiple heterochronic parameters translate into large phenotypic differences in feeding morphology.     

Feedbacks, thresholds and synergies in global change: population as a dynamic factor

The role of feedbacks, thresholds, and synergies in environmental science, and their implications for environmental degradation under a growing human population, are reviewed. A detailed analysis of the impacts of climate change on water resources is used to elucidate mechanisms by which nonlinearities arise in environmental science. Additional examples are drawn from analysis of soil degradation and non-climate related degradation of water resources. The often-assumed notion that impacts will grow in proportion to population size is shown to be overly optimistic. In particular, feedbacks, thresholds, and synergies among multiple threats, tend to amplify risk and cause environmental impacts to grow considerably faster than linearly in population size, even when the per-capita living standard and the technological systems deployed to achieve that living standard are assumed to remain constant.     

Developmental differences in visceral morphology of megophryine pelobatid tadpoles in relation to their body form and mode of life

Tadpoles face severe packing constraints on viscera within the pleuro-peritoneal cavity because of their extremely short torsos—a feature they share with adult anurans—and the concomitant need for relatively slender torsos for efficient locomotion. We examined the effects of differences in body form and habits on the size, shape and development of viscera in three kinds of sympatric, stream-associated pelobatid tadpoles. Leptobrachium montanumlarvae are generalized, wide, deep-bodied tadpoles. Larval Leptolalax gracilis are very slender and live in the crevices between rocks on the bottom of riffles. Larval Megophrys nasuta are intermediate between the other two in body form, and live with L. montanum in a variety of microhabitats but feed at the surface film. In all three species, liver, gall bladder, arid kidneys begin development early and grow isometrically throughout larval life. The gut and pancreas have a growth spurt shortly after hatching, then grow at a constant rate until near metamorphosis when both shrink drastically. The spleen grows at a slower rate than the body throughout the larval period. Lungs do not appear in L. gracilis until the tadpole approaches metamorphosis, which accords with its benthic habits, whereas they grow throughout the larval period in L. montanum and M. nasuta. In M. nasuta, however, the lungs are unusually wide anteriorly; this shifts buoyancy forward and facilitates the head-up feeding posture characteristic of that species. Gonads appear early in L. montanum and L. gracilis, but not until near metamorphosis in M. nasuta. We suggest that accelerated gonadal development in tadpoles characterizes species that metamorphose close to their size at first reproduction. Leptobrachium montanum, with the bulkiest body and most generalized habits, has relatively and absolutely the largest gut, liver (x of combined gut and liver volume = 24%, of total volume), and kidneys. Leptolalax gracilis, the most slender tadpole, has relatively the smallest combined gut and liver volume (x = 10% of total volume). Other premetamorphic differences among the species were observed in gut coiling, liver, pancreas and kidney shape and left/right asymmetry of urogenital organs. The major interspecific differences we observed in the size, shape, and developmental patterns of viscera in tadpoles are clearly related to interspecific differences in torso shape, microhabitat distribution and mode of feeding.     

Bud-Localization of CLB2 mRNA Can Constitute a Growth Rate Dependent Daughter Sizer

Maintenance of cellular size is a fundamental systems level process that requires balancing of cell growth with proliferation. This is achieved via the cell division cycle, which is driven by the sequential accumulation and destruction of cyclins. The regulatory network around these cyclins, particularly in G₁, has been interpreted as a size control network in budding yeast, and cell size as being decisive for the START transition. However, it is not clear why disruptions in the G₁ network may lead to altered size rather than loss of size control, or why the S-G₂-M duration also depends on nutrients. With a mathematical population model comprised of individually growing cells, we show that cyclin translation would suffice to explain the observed growth rate dependence of cell volume at START. Moreover, we assess the impact of the observed bud-localisation of the G₂ cyclin CLB2 mRNA, and find that localised cyclin translation could provide an efficient mechanism for measuring the biosynthetic capacity in specific compartments: The mother in G₁, and the growing bud in G₂. Hence, iteration of the same principle can ensure that the mother cell is strong enough to grow a bud, and that the bud is strong enough for independent life. Cell sizes emerge in the model, which predicts that a single CDK-cyclin pair per growth phase suffices for size control in budding yeast, despite the necessity of the cell cycle network around the cyclins to integrate other cues. Size control seems to be exerted twice, where the G₂/M control affects bud size through bud-localized translation of CLB2 mRNA, explaining the dependence of the S-G₂-M duration on nutrients. Taken together, our findings suggest that cell size is an emergent rather than a regulatory property of the network linking growth and proliferation.     

Effect of canopy size and shape on the tuber yield of sixteen potato genotypes

An experiment was conducted to evaluate the utility of visual assessment of canopy characteristics in the selection of high-yielding clones for the southern Canadian Prairies. Over a 3-yr period the yield development characteristics of 16 genotypes, selected to represent a range of canopy types, both in size and shape, were studied by destructive sampling at five stages during the growing season. While the response over harvest dates was not consistent for the canopy size or shape groups for most yield variables, it was demonstrated that under the short season growing conditions in southern Alberta, canopies intermediate in size and degree of upright growth habit appear to be most efficient in terms of maximising tuber bulking rates. It was concluded that for the southern Canadian Prairies, selection of genotypes with these types of canopy structure is consistent with the objective of developing early-maturing cvs with high tuber yield.     

Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens: responses of the wild-type

Primary chloronemata growing from germinated spores of the moss Physcomitrella patens adopt one of two preferred polarotropic orientations depending on the wavelength and photon fluence rate of monochromatic light. Growth is mainly parallel to the electrical vector of plane polarised light in blue light and higher fluence rates of red light, and perpendicular to the electrical vector in the green and far-red regions of the spectrum and in low fluence rates of red light. The transition between the two polarotropic orientations, at wavelengths where it can be observed, usually occurs over a narrow range of fluence rates, and at this point the filaments do not grow randomly but tend to adopt in approximately equal numbers one of the preferred directions of growth. The primary chloronemata are positively phototropic in far-red light and in red light of low fluence rates, but tend to grow at right angles to the incident light in high fluence rates of red light. Simultaneous illumination with a high fluence rate of red light and a low fluence rate of far-red light causes a marked increase in the percentage of filaments growing towards the red light source at the expense of those growing at right angles to it, supporting the hypothesis that in red and far-red light, at least, the responses are controlled by the photoequilibrium of a phytochrome pool.