Insights into the molecular basis of thermal stability from the structure determination of Pyrococcus furiosus gluatamate dehydrogenase

Abstract: The structure determination of the glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus has been completed at 2.2 Å resolution. The structure has been compared with the glutamate dehydrogenases from the mesophiles Clostridium symbiosum, Escherichia coli and Neurospora crassa . This comparison has revealed that the hyperthermophilic enzyme contains a striking series of networks of ion-pairs which are formed by regions of the protein which contain a high density of charged residues. Such regions are not found in the mesophilic enzymes and the number and extent of ion-pair formation is much more limited. The ion-pair networks are clustered at both inter domain and inter subunit interfaces and may well represent a major stabilising feature associated with the adaptation of enzymes to extreme temperatures.     

Genetically stable cell lines of cucumber for the large-scale production of diploid somatic embryos

For the initiation of embryogenic cucumber ( Cucumis sativus L.) cell lines, from excised radicles, directly in liquid medium, the culture regime, explant density and type and concentration of hormones were adjusted so that pro-embryogenic masses (PEMs) were formed within about 8 weeks. The established cucumber cell lines were maintained tor several years without loss of embryogenic and genetic stability. The ploidy level of somatic embryos from different cucumber eell lines was either diploid or tetraploid and depended on the ploidy level of Ihe cell line. Cucumber cell lines that produced only diploid embryos were obtained by selecting completely diploid explant material and growing it in the dark during the initiation phase. Mixoploid explains could lead to tetraploid or mixoploid ceil lines. Isolation and additional selection and subculturing of single PEMs resulted in either completely diploid or tetraploid cell lines, indicating that all cells of individual PEMs are either diploid or tetraploid. The ernbryogenic cucumber cell Imes, differing only in ploidy level, were indistinguishable in growth rate and embryogetiic potential and were genetically stable over several years.     

Evolutionary dynamics of predator-prey systems: an ecological perspective

 Evolution takes place in an ecological setting that typically involves interactions with other organisms. To describe such evolution, a structure is needed which incorporates the simultaneous evolution of interacting species. Here a formal framework for this purpose is suggested, extending from the microscopic interactions between individuals – the immediate cause of natural selection, through the mesoscopic population dynamics responsible for driving the replacement of one mutant phenotype by another, to the macroscopic process of phenotypic evolution arising from many such substitutions. The process of coevolution that results from this is illustrated in the context of predator–prey systems. With no more than qualitative information about the evolutionary dynamics, some basic properties of predator–prey coevolution become evident. More detailed understanding requires specification of an evolutionary dynamic; two models for this purpose are outlined, one from our own research on a stochastic process of mutation and selection and the other from quantitative genetics. Much of the interest in coevolution has been to characterize the properties of fixed points at which there is no further phenotypic evolution. Stability analysis of the fixed points of evolutionary dynamical systems is reviewed and leads to conclusions about the asymptotic states of evolution rather different from those of game-theoretic methods. These differences become especially important when evolution involves more than one species. Received 10 November 1993; received in revised form 25 July 1994     

Varietal mixtures: a viable strategy for sustainable productivity in subsistence agriculture

Remarkable parallels link the development of varietal mixtures across subsistence farming systems. Mixtures are grown and persist because they prolong harvest and income flow and provide diversity of diet. From our review of research on agronomic and disease aspects of mixtures in modern agriculture, it is also clear that improved stability and decreased disease severity are common features of mixtures relative to their components in monoculture. Such advantages are of value to both modern and subsistence agriculture. However, in the majority of cases, the yield advantage of mixtures is small. Overall, we conclude that varietal mixtures are presently a viable strategy for sustainable productivity in subsistence agriculture, have potential for improvement without sacrifice of diversity, are an important resource for future global food production and may have an expanding role in modern agriculture in situations where qualitative uniformity is not the guiding priority.     

The effect of a propionic acid bacterial inoculant applied at ensiling on the aerobic stability of wheat and sorghum silages

The effect of a new strain ofPropionibacterium shermanii (PAB), applied at ensiling, on the aerobic stability of wheat and sorghum silages was studied in several experiments under laboratory conditions. In the one experiment with wheat and in those with sorghum a lactic acid bacteria (LAB) inoculant (Lactobacillus plantarum andPediococcus cerevisiae) was also included. After treatment, the chopped forages were ensiled in 1.5-L anaerobic jars which were sampled in triplicate on predetermined dates to follow fermentation dynamics. At the end of the experiments, the silages were subjected to an aerobic stability test. The PAB inoculant improved the aerobic stability only in one experiment with wheat, in which the decrease in pH was very slow; the final pH remained relatively high (4.5). The PAB-treated silages contained 19.5±2.0 g of propionic acid per kg of dry matter. In the experiments with sorghum, the control and PAB-inoculated silages were stable, whereas LAB-inoculated silages deteriorated. The results suggest that PAB can survive in and improve the aerobic stability of only slow-fermenting silages which are prone to aerobic deterioration.     

Lactam bridge stabilization of α-helical peptides: Ring size,orientation and positional effects

A series of 14 residue amphipathic α-helical peptides, in which the sidechains of glutamic acid and lysine have been covalently joined, was synthesized in order to determine the effect of spacing, position and orientation of these lactam bridges. It was found that although an (i, i+3) spacing would position the lactam bridge on the same face of the helix, these lactams with 18-member rings were actually helix-destabilizing regardless of position or location. On the other hand, (i, i+4) lactams with 21-member rings were helix-stabilizing but this was dependent on orientation. Glutamic acid-lysine lactams increased the helical content of the peptide when compared with their linear homologue in benign conditions (50 mM KH₂PO₄, 100 mM KCl, pH 7). Two Glu-Lys (i, i+4) lactams located at the N- and C-termini gave rise to a peptide with greater than 99% helical content in benign conditions. Peptides with Lys-Glu oriented lactams were random structures in benign conditions but in the presence of 50% TFE could be induced into a helical conformation. The stability of the single-stranded α-helices, as measured by thermal denaturations in 25% TFE indicated that Glu-Lys oriented lactam bridges stabilized the helical conformation relative to the linear unbridged peptide. One Glu-Lys lactam in the middle of the peptide was more effective at stabilizing helical structure than two Glu-Lys lactams positioned one at each end of the molecule. The lactams with the Lys-Glu orientation were destabilizing relative to the unbridged peptide. This study demonstrates that correct orientation and position of a lactam bridge is critical in order to design peptides with high helical content in aqueous media.     

Modelling the effect of treatment and behavioral change in HIV transmission dynamics

In this paper we analyze a model for the HIV-infection transmission in a male homosexual population. In the model we consider two types of infected individuals. Those that are infected but do not know their serological status and/or are not under any sort of clinical /therapeutical treatment, and those who are. The two groups of infectives differ in their incubation time, contact rate with susceptible individuals, and probability of disease transmission. The aim of this article is to study the roles played by detection and changes in sexual behavior in the incidence and prevalence of HIV. The analytical results show that there exists a unique endemic equilibrium which is globally asymptotically stable under a range of parameter values whenever a detection /treatment rate and an indirect measure of the level of infection risk are sufficiently large. However, any level of detection/ treatment rate coupled with a decrease of the transmission probability lowers the incidence rate and prevalence level in the population. In general, only significant reductions in the transmission probability (achieved through, for example, the adoption of safe sexual practices) can contain effectively the spread of the disease.     

Predictions for oxygen supply control to enhance population stability of engineered production strains

The simultaneous growth and product formation in a microbial culture is an important feature of several laboratory, industrial, and environmental bioprocesses. Metabolic burden associated with product formation in these bioprocesses may lead to growth advantage of a nonproducing mutant leading to a loss of the producing population over time. A simple population dynamics model demonstrates the extreme sensitivity of population stability to the engineered productivity of a strain. Here we use flux balance analysis to estimate the effects of the metabolic burden associated with product secretion on optimal growth rates. Comparing the optimal growth rates of the producing and nonproducing strains under a given processing condition allows us to predict the population stability. In order to increase stability of an engineered strain, we determine processing conditions that simultaneously maximize the growth rate of the producing population while minimizing the growth rate of a nonproducing population. Using valine, tryptophan, and lysine production as specific examples, we demonstrate that although an appropriate choice of oxygenation may increase culture longevity more than twofold, total production as governed by economic criterion can be increased by several orders of magnitude. Choice of optimal nutrient and oxygen supply rates to enhance stability is important both for strain screening as well as for culture of engineered strains. Appropriate design of the culture environment can thus be used to enhance the productivity of bioprocesses that use engineered production strains. (c) 1994 John Wiley & Sons, Inc.