Catabolite regulation of antibiotic biosynthesis

Conclusion In view of the possible mediation of carbon catabolite repression of antibiotic biosynthesis by phosphorylated substances, the concept of the role of phosphorus in the regulation of secondary metabolism should be re-evaluated. Many conclusions are based on an analogy with the effect of phosphorus in animal or plant cells (for review cf. Martin 1977). However, in contrast with plant and animal physiology, the production physiology of actinomycetes, typical soil microorganisms, was studied under nonphysiological conditions of nutrient over-supply in a submerged culture. This is analogous e.g. to space biology which studies the physiology of organisms in the state of weightlessness. Both disciplines have an extremely high significance but the elucidation of basic biological regularities is to be carried out back on Earth. In conclusion we may state that a critical evaluation of our contemporary knowledge seem to support the hypothesis that the antibiotic biosynthesis in actinomycetes, as well as the spore formation in bacilli — both regulated by the mechanism of catabolite repression — has its ecological significance, i.e. makes it possible for the organism to survive under conditions when vegetative growth is limited. For this reason this aotivity was preserved in the course of evolution as a protective mechanism of microbial populations and remained a part of the genome of many species.     

Metabolism characteristics of <Emphasis Type="Italic">Chelativorans oligotrophicus</Emphasis> by two-phase growth on the mixture of EDTA and glucose

The obligate destructor of ethylene diamine tetraacetate—a culture of Chelativorans oligotrophicus LPM-4—did not grow on a medium with glucose, but it was good to use it under cultivation on a mixture with EDTA after considerable decrease of the EDTA concentration in the medium (two-phase growth). Strong inhibition of hexokinase and glucose 6-phosphate dehydrogenase in cell exracts 4 mM EDTA was revealed. Using EDTA, cells accumulated polyphosphates whose rate decreased during glucose utilization phase. High activities of polyphosphate biosynthesis ferments (adenylat kinase and polyphosphate kinase) were distinguished during the first phase of the cultivation; considerable decrease of them and increase of polyphosphate glucokinase were found during the second phase of the cultivation. This points to the possible participating of polyphosphates in glucose metabolism as a supplementary energy source.     

Art in global context: An evolutionary/functionalist perspective for the 21<Superscript>st</Superscript> century

Regarding the arts as something peopledo — as behaviors, rather than the residue or artifacts of behavior — makes possible a theoretical grounding about their nature and importance, an endeavor that current anthropology of art has largely abandoned. A reconsideration of the suspect and largely discarded terms “functionalism” and “evolutionism” is presented in light of current evolutionary thinking. It is suggested that a contemporary reformulation of these concepts, illustrated by the author's Darwinian or “adaptationist” perspective on art, supports aims and claims of current anthropology of art, and contributes new focus and direction to its endeavors.     

Singularity solution of Lanir's osmoelasticity: verification of discontinuity simulations in soft tissues

The literature characterizes cartilaginous tissues as osmoviscoelastic. Understanding the damage and failure of these tissues is essential for designing treatments. To determine tissue strength and local stresses, experimental studies—both clinical and animal—are generally supported by computational studies. Verification methods for computational studies of ionized porous media including cracks are hardly available. This study provides a method for verification and shows its performance. For this purpose, shear loading of a finite crack is addressed analytically and through a commercial finite element code. Impulsive shear loading by two-edge dislocation of a crack was considered in a 2D plane strain model for an ionized porous medium. To derive the analytical solution, the system of equation is decoupled by stress functions. The shear stress distribution at the plane of the crack is derived using Fourier and Laplace transformations. The analytical solution for the shear stress distribution is compared with computer simulations in ABAQUS version 6.4-5. Decoupling of the equations makes it possible to solve some boundary value problems in porous media taking chemical effects into account. The numerical calculations underestimate the shear stress at the crack-tips. Mesh refinement increases accuracy, but is still low in the neighborhood of the crack-tips.     

High-level production of a kringle domain variant by high-cell-density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human kringle domains (KDs) are ubiquitously expressed binding modulators that fold into seven flexible loops and it has been previously demonstrated that KDs can be engineered toward target-specific binding proteins as a non-antibody protein scaffold. Here, we report a method for efficient expression of a KD derivative (KD548)—a promising anti-cancer agent—by high-cell-density culture of Escherichia coli at a preparative scale production. The correct folding of KD548 requires three disulfide bonds. Nevertheless, cytoplasmic expression of KD548 in E. coli led to good yields of highly soluble proteins with high activity. For efficient expression, four sets of expression systems consisting of different promoters (lac or T7) and fusion tags (His or FLAG) were examined. Of these, the expression system using a combination of the T7 promoter with the FLAG tag resulted in the highest production in shake flask cultivation as well as in high-cell-density cultivation performed in a 6.6-L jar bioreactor. When protein expression was induced at high-cell density (optical density [OD] = 100) and when complex feeding solutions were supplemented, cell density (maximum OD = 184) and production yield (∼5.4 g/L) were significantly enhanced to values that were much higher than those found previously with Pichia cultivation (<8 mg/L).     

Study of the reciprocal relationship of the exoantigens ofClostridium perfringens type A in the culture medium and the bacterial cells during cultivation

The times of the active release ofClostridium perfringens antigens—lecithinase, collagenase and alkaline proteinase—were determined. It was found that lecithinase release attained maximum values after 4–9 hours' cultivation of the microorganisms and stopped completely after 24–36 hours. The course of collagenase release was the same. Maximum proteinase release was determined within 36 to 48 hours' cultivation ofClostridium perfringens. Preformed lecithinase disappeared very rapidly from the culture medium. The presence of the components of the exoantigen complex was determined in the bacterial cell cytoplasm by the immunoadsorption method. Their distribution in the fractions isolated by differential high speed centrifugation of homogenates of disintegrated bacterial cells was studied. Their ribosomal fraction, represented by four groups of ribosomes with the maximum sedimentation coefficients 110 and 130S, contains alkaline proteinase. Its activity diminishes during cultivation of the microorganism and proteolytic activity accumulates in the hyaloplasm. The absence of lecithinase and collagenase in the bacterial cells was demonstrated. The plate will be found at the end of the issue.     

Analysis of the exponential decay model of the neuron showing frequency threshold effects

The exponential decay model of a neuron has been analyzed using the “random walk” approach of stochastic processes and an “absorbing barrier” solution is obtained forg ^T (s)—the Laplace transform of the output pulse interval density function. An expression for the mean output frequency is derived from this and a variety of input-output curves plotted which show frequency threshold effects in single neurons. Our results are compared with those of other authors obtained by computer simulation techniques, and the significance of these results discussed with reference to the possible behavior of networks constructed of such neuron units.     

An improved system of subjecting plants to water stress

An improved system of plant cultivation at stable and specific levels of polyethyleneglycol (PEG, mol. mass 1400–1600) — induced water stress has been described. To set up this system a perforated tubular glass vessel containing soil to support seedling growth was wrapped externally first with a layer of macroporous silica gel-G and then with three layers of a dialysis membrane of a lower exclusion limit (2000 mol. mass). Effects of 8 days of PEG — induced stress have been studied on uptake and translocation of N and P and growth of barley (Hordeum vulgare L. cv. KN 16) seedlings. Some of the noteworthy improvements of the system were exelusion of PEG from the plant consequent upon use of silica gel-membrane combination, shorter time (2 days) for the soil-plant-air continuum to attain steady state, and stability of the plant water potential over a period of a few days.     

Oligomeric state investigation of flavocytochrome CYP102A1 using AFM with standard and supersharp probes

Atomic force microscopy with two types of probes—standard (radius of curvature R ∼ 10 nm) and supersharp (R ∼ 2 nm)—was used to determine an oligomeric state of CYP102A1. Using the standard probes CYP102A1 images were obtained in liquid, air and vacuum environments, and a CYP102A1 monomer: oligomer ratio α ≈ 1 was also determined. However, the use of standard probes did not allow to resolve structures of these oligomers. Using the supersharp probes it was possible to determine not only the monomer: oligomer ratio, but also to evaluate the dimer: trimer: tetramer ratio in vacuum. Thus, the ratio α for CYP102A1 in liquid can be determined by the standard probes in liquid, air, and vacuum, while oligomeric states of this protein can be specified by using the supersharp probes in vacuum.     

Question 3: The Problem of Macromolecular Sequences: The Forgotten Stumbling Block

The Author agrees in principle with the question/statement, but states also that an important qualification is needed within this question. In fact, it is not possible by the bottom up approach to find the conditions for the synthesis of our actual proteins—lysozyme, chymotrypsin or the like—however it is possible to show experimentally that co-oligopeptides chains of that length can be produced by prebiotic reactions. Considering such a synthesis, it is important to recall that proteins—and nucleic acids—are not simply polymers, but are co-polymers, and the kinetics and thermodynamics attending the synthesis of copolymers poses stringent constraints for the biogenesis and growth of specific sequences. Such constraints are examined and discussed. 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.     

Consciousness is Cheap, Even if Symbols are Expensive; Metabolism and the Brain’s Dark Energy

Use of symbols, the key to the biosemiotics field as to many others, required bigger brains which implied a promissory note for greater energy consumption; symbols are obviously expensive. A score years before the current estimate of 18–20% for the human brain’s metabolic demand on the organism, it was known that neural tissue is metabolically dear. This paper first discusses two evolutionary responses to this demand, on both of which there is some consensus. The first, assigning care of altricial infants with burgeoning brains (and in human infants the metabolic demand peaks at 65% of the total) to “allomothers” is not unique to humans. The second, using relatively small neurons as primates do, risks misfires past a certain minimal value. Moreover, in apparent paradox, there is an increasing consensus that large “Von Economo” neurons are critical for communication. This paper’s main contribution is the discussion of two further evolutionary tricks. The first is the use of self-similarity in the cortex, both in structure and process, to allow the cortex readily—and in energetic terms, parsimoniously—to shift between states in a high-dimensional space. This leads to discussion of the kind of formalism appropriate to model these shifts, a formalism which—it is tentatively suggested—may do double duty for the modeling of symbolic thought. The second trick is the superimposition on the background “white noise” of neural firing of EEG-detected waves like gamma. The paper describes a method, using the Hilbert transform, of calculating the dips in energy consumption as the brain is transitioned by gamma waves. It is hypothesized that consciousness may be a spandrel, the incidental result of a neurodynamic imperative that the brain enter a maximally sensitive (in sensory terms) “zero power” state a few times a second. If that is the case, then there are obvious benefits for health in meditation, which can be viewed as a state of consciousness extended over time by limiting afferent stimuli.     

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the calgary biofilm device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.     

Agar gel immobilization ofBacillus brevis cells for production of thermostable α-amylase

Growing cells of a thermophilic strain ofBacillus brevis, producer of thermostable α-amylase, were immobilized by entrapment in agar gel. Optimum immobilization conditions for effective α-amylase production in batch fermentations were established (gel concentration 3%, initial biomass concentration in the gel 0.8% (W/V), and preculture age—late exponential phase). The dynamics of α-amylase synthesis by the biocatalysts obtained under the optimal conditions was compared with that of free cells and the operational stability of the biocatalysts was studied in semicontinuous cultivation experiments. Maximum α-amylase yields (252% of the control) were achieved after the second cycle of cultivation. Scanning electron microscopy was used to characterize the bacteria entrapped in agar gel.     

Two-step concerted mechanism for alkane hydroxylation on the ferryl active site of methane monooxygenase

 A two-step concerted mechanism for the conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO) is discussed. We propose that the enzymatic reaction mechanism is essentially the same as that of the gas-phase methane-methanol conversion by the bare FeO⁺ complex. In the initial stage of our mechanism, the ferryl (Fe—O) "iron" active site of intermediate Q and substrate methane come into contact to form the initial Q (CH₄) complex with an OFe—CH₄ bond. The C—H bonds of methane are significantly weakened by the formation of a five-coordinate carbon species, through orbital interactions between a C _3v - or D _2d -distorted methane and the Fe—O active site. The important transition state for an H atom abstraction exhibits a four-centered structure. The generated intermediate involves an HO—Fe—CH₃ moiety, and it is then converted into the final product complex including methanol as a ligand through a methyl migration that occurs via a three-centered transition state. The two-step concerted mechanism is consistent with recent experiments on regioselectivity of enzyme-catalyzed alkane hydroxylations. Received: 15 September 1997 / Accepted: 20 December 1997     

Engineering Human Cooperation

In a laboratory experiment, we use a public goods game to examine the hypothesis that human subjects use an involuntary eye-detector mechanism for evaluating the level of privacy. Half of our subjects are “watched” by images of a robot presented on their computer screen. The robot—named Kismet and invented at MIT—is constructed from objects that are obviously not human with the exception of its eyes. In our experiment, Kismet produces a significant difference in behavior that is not consistent with existing economic models of preferences, either self- or other-regarding. Subjects who are “watched” by Kismet contribute 29% more to the public good than do subjects in the same setting without Kismet.     

A two-stage solar photobioreactor for cultivation of microalgae based on solar concentrators

A novel two-stage experimental photobioreactor (PBR) with a total volume of 450 L and based uniquely on solar concentrators—linear Fresnel lenses—has been constructed and tested. Daily courses of irradiance, and also its distribution inside cultivation tubes, were studied in two unit types. The supra-high irradiance units in the ‘roof’ achieved a maximum summer value above 6 mmol photon m⁻² s⁻¹, while irradiance in the vertical-facade units was lower than ‘ambient’. In model cultivations, cultures of the cyanobacterium Arthrospira platensis were cultivated at much higher solar irradiances than those usually recorded outdoors in summer, indicating that this organism is resilient to high-irradiance (photoinhibition). Starting from a biomass density of 0.5 g L⁻¹ at optimum temperature, the cultures grew exponentially. A two-stage cultivation process of the green microalga Haematococcus pluvialis was investigated with respect to correlations between photochemical activities and astaxanthin production. The culture was first grown in low-irradiance units, and then exposed to supra-high irradiance when the rate of astaxanthin production was 30–50% higher than in the culture exposed to ‘ambient’ irradiance. Within 4 days, the astaxanthin content reached 3% of dry weight, whereas under ambient irradiance the astaxanthin content was 25% lower.     

Differences in functional activity of cultivated human vascular endothelium cells derived from various donors

The endothelia of blood vessels fulfill multiple functions in an organism under physiological or pathological conditions. It is possible to model the processes that take place in the vascular endothelium on cultivated endothelial cells. Unlike permanent cell lines, research on primary cell cultures may lead to inconsistent results. In this work, endothelium cultures derived from umbilical cords of 20 donors have been compared for markers that characterize the functional activities of endothelium. It was found that, after 3 h in culture, it was possible to divide cultures into two groups, i.e., high — and low-expressing markers. An analysis of cytokines showed that the level of spontaneous production of IL-1 β in groups did not vary in 24 and 48 h, whereas levels of IL-6 and IL-8 production were increased at 24 and 48 h, but the difference between groups was not evident. TNFα production during cultivation was only increased in the low-expressing group. The amount of sP-selectin and sE-selectin in cultural medium was only enhanced in low-producing cultures, whereas the increase in sICAM-1 during cultivation was observed in highly productive cultures. The increase in sPECAM-1 was revealed in both high- and low-reproducing cultures and the differences between the groups were retained. The level of sVE-cadherin in the culture medium remained unchanged throughout cell cultivation. The levels of nitrite in the culture medium, which reflect the amount of NO, was augmented in all cultures and the difference between the groups was retained. The concentration of endothelin-1 was increased; however, in culture media of various cultures, its amounts were similar; therefore, it was not possible to create groups that reflect the level of its production. The level of von Willebrand factor in culture medium was increased during the cultivation of both groups; however, the difference between groups was lost. The level of matrix metalloproteinase-1 in culture medium was increased during cell cultivation.     

Preparation of protoplasts and regeneration of intact cells ofStreptomyces cinnamonensis

Protoplasts were prepared and intact cells were regenerated inStreptomyces cinnamonensis— a monensin producer— to make genetic manipulations with this strain possible. 70–80% of protoplasts were formed and up to 90% of them could regenerate into intact cells.     

Concept formation in neural networks: Implications for evolution of cognitive functions

Parallel systems composed of many interconnected elements are both simple brain models and possible novel computer architectures. Potential advantages of such systems are massive parallelism with resulting speedup of computation as well as general ability to compute with noisy, corrupted, or missing data. Parallel, distributed, associative models have pronounced psychologies. Some ways of handling information are natural for them, and some things that we might want them to do are unnatural and quite difficult to do. A question of considerable interest is whether the models’ capabilities and limitations are features of human psychology. Such systems form categories based on the structure their inputs and display behavior that looks as if they form and use simple concepts. However, if noisy examples are learned, an initially stable concept structure may break up. One very simple function of names attached to categories — i.e. a rudimentary language — could be to stabilize a concept structure against fragmentation. In addition, if the statistical structure of the names reflects the statistical structure of the inputs, capacity and reliability of categorization and recognition is enhanced.     

Quasi steady state growth of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in glucose-limited acceleration stat (A-stat) cultures

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h⁻² after initial stabilization of the cultures in chemostat at D = 0.2–0.3 h⁻¹. It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used—at an acceleration rate 0.003 h⁻² the quasi steady state growth was observed until μ _crit = 0.59 h⁻¹, which is also the μ _max value for the culture. Lower values of μ _crit were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h⁻¹ was immediately disrupted after initiating acceleration at the highest acceleration rate studied—0.06 h⁻². Observation was made that differences [Δ(μ − D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h⁻² (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate—it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S ₀ = 5 g L⁻¹ glucose instead of S ₀ = 10 g L⁻¹. It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility—Δ(μ − D) was less than 5%, standard deviations of biomass yields per ATP produced (Y _ATP), and biomass yields per glucose consumed (Y _XS) were less than 15%.