Immunological adjuvants efficiently induce antigen-specific T cell responses in old mice: implications for vaccine adjuvant development in aged individuals

The morbidity and mortality of infectious diseases are significantly increased in aged humans. Hence, vaccination has been suggested as a means to reduce or prevent the impact of infections on old individuals. However, it has remained unresolved whether or not standard vaccine adjuvants such as aluminum hydroxide (Alum) are similarly efficacious in old individuals, as compared to young adults. Here, we have investigated the effects of prototypic immunological adjuvants, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), or Alum on HEL-specific T cell responses in young adult and old mice. We report that independent of the adjuvant used, the induced T cell responses to the prototypic protein antigen hen eggwhite lysozyme (HEL) were similar in young adult and old mice in terms of cytokine production, T cell frequencies, determinant specificity, and T cell repertoire. The results suggest that vaccine adjuvants developed in young adults should be equally effective in inducing T cell immunity in old individuals.     

Reconciling genetic expectations from host specificity with historical population dynamics in an avian brood parasite, Horsfield's Bronze-Cuckoo Chalcites basalis of Australia

Mitochondrial DNA (mtDNA) is being used increasingly to explore the evolution of host specificity in avian brood parasites. A stable coevolutionary equilibrium between multiple phylogenetically unrelated hosts and a brood parasitic species predicts that mtDNA diversity in the parasite should be relatively deep and phylogenetically structured. Also, the different intraspecific clades resulting from parasitism to multiple sympatric hosts should themselves occur sympatrically. However, mtDNA diversity in brood parasites is as susceptible to effects of historical population dynamics as in any species. We demonstrate the relevance of these dynamics to the use of mtDNA in understanding coevolution between an Australian brood-parasite, Horsfield's Bronze-Cuckoo Chalcites basalis and its hosts, Malurus fairy-wrens and Acanthiza thornbills. Previous ecological and behavioural analyses argue that Malurus- and Acanthiza-specific host races exist in C. basalis. Yet mtDNA diversity in C. basalis is low and phylogenetically unstructured (mean sequence divergence 0.15 +/- 0.07%, range 0.00%-0.31%) and tests of mtDNA neutrality and range expansion vs. population stability (Tajima's D, Fu & Li's F* and D*, Fu's F(S), mismatch analyses) all indicate that C. basalis has expanded its range very recently, probably within the last few tens of thousands of years following climatic amelioration after a peak of aridity in the late Pleistocene. The low mtDNA diversity and its lack of phylogenetic structure in C. basalis deny the existence of evolutionarily long-term stable host races in C. basalis but not the possibility of recently evolved ones. They highlight the need for renewed behavioural and ecological study of the relationship between C. basalis and its hosts. Our findings illustrate the need to understand the evolutionary context in which a brood parasite and its hosts have evolved if mtDNA data are to be used in testing hypotheses concerning the origin and maintenance of host specificity. They also add to the growing body of work illustrating the use of mismatch analyses and Fu's F(S) in detecting range expansions.     

Crystal doping aided by rapid expansion of supercritical solutions

The purpose of this study was to test the utility of rapid expansion of supercritical solution (RESS) based cocrystallizations in inducing polymorph conversion and crystal disruption of chlorpropamide (CPD). CPD crystals were recrystallized by the RESS process utilizing supercritical carbon dioxide as the solvent. The supercritical region investigated for solute extraction ranged from 45 to 100°C and 2000 to 8000 psi. While pure solute recrystallization formed stage I of these studies, stage II involved recrystallization of CPD in the presence of urea (model impurity). The composition, morphology, and crystallinity of the particles thus produced were characterized utilizing techniques such as microscopy, thermal analysis, x-ray powder diffractometry, and high-performance liquid chromatography. Also, comparative evaluation between RESS and evaporative crystallization from liquid solvents was performed. RESS recrystallizations of commercially available CPD (form A) resulted in polymorph conversion to metastable forms C and V, depending on the temperature and pressure of the recrystallizing solvent. Cocrystallization studies revealed the formation of eutectic mixtures and solid solutions of CPD+urea. Formation of the solid solutions resulted in the crystal disruption of CPD and subsequent amorphous conversion at urea levels higher than 40% wt/wt. Consistent with these results were the reductions in melting point (up to 9°C) and in the ΔH_fvalues of CPD (up to 50%). Scanning electron microscopy revealed a particle size reduction of up to an order of magnitude upon RESS processing. Unlike RESS, recrystallizations from liquid organic solvents lacked the ability to affect polymorphic conversions. Also, the incorporation of urea into the lattice of CPD was found to be inadequate. In providing the ability to control both the particle and crystal morphologies of active pharmaceutical ingredients, RESS proved potentially advantageous to crystal engineering. Rapid crystallization kinetics were found vital in making RESS-based doping superior to conventional solvent-based cocrystallizations.     

Toward minimalist models of larger proteins: a ubiquitin-like protein

Our recently developed off-lattice bead model capable of simulating protein structures with mixed alpha/beta content has been extended to model the folding of a ubiquitin-like protein and provides a means for examining the more complex kinetics involved in the folding of larger proteins. Using trajectories generated from constant-temperature Langevin dynamics simulations and sampling with the multiple multi-histogram method over five-order parameters, we are able to characterize the free energy landscape for folding and find evidence for folding through compact intermediates. Our model reproduces the observation that the C-terminus loop structure in ubiquitin is the last to fold in the folding process and most likely plays a spectator role in the folding kinetics. The possibility of a productive metastable intermediate along the folding pathway consisting of collapsed states with no secondary structure, and of intermediates or transition structures involving secondary structural elements occurring early in the sequence, is also supported by our model. The kinetics of folding remain multi-exponential below the folding temperature, with glass-like kinetics appearing at T/T(f) approximately 0.86. This new physicochemical model, designed to be predictive, helps validate the value of modeling protein folding at this level of detail for genomic-scale studies, and motivates further studies of other protein topologies and the impact of more complex energy functions, such as the addition of solvation forces.     

Selective multinationality how environmental management helps high-tech SMES identify high-growth,low-risk markets

Background  Small and medium sized enterprises in developed regions, as well as larger ‘Southern’ firms are more exposed to risk in regards to allocation decisions. This includes expenditures on fixed assets, R&D and environmental management, as well as the choice of suitable markets for the product or service at hand. For either type of firm, the consequences of not selecting, or timing, suitable markets can lead to bankruptcy. Objectives  This paper examines means for SMEs to expand internationally given the current fiscal climate, and rapid advances in certain enabling technologies. The goal is to identify a set of criteria which smaller firms can use to globalize which reduce risk and increase access to capital. Methods  Case studies are presented of a European Start-Up as well as a century-old Latin American firm, both in the water-treatment sector. The similarities in the risk-reward profiles are used to identify some key strategic guidelines for enterprises wishing to be, selectively, multinational. Results and Discussion  Selective multinationality is an immediate plan for international expansion which identifies the most appropriate markets, independent of their proximity to the firm’s production base or headquarters. At the base of selective multinationality, is the goal to impose the firm’s product in regions of high growth, high selling price and low risk of price dumping. Therefore, this requires theto resist the temptation to expand to markets based on merely geographical proximity. The globalization strategy should be established, along with environmental management, from the outset of the creation of the firm. It will be shown, herein, to be a key driver in the valuation of High Tech industrial SMEs, in particular those with high risk-high reward tradeoffs. Selective multinationality can best be implemented if the firm in question has a technology which addresses unmet needs in niche markets, where there is an expanding customer base focusing on client relationships and cost effectiveness, and where the multinationals enter from outside the sector via resellers. It will be demonstrated to be effective for startups from developed regions, as well as SMEs from developing/emerging countries, to both of whom the consequences of sub-optimal resource allocation can risk the future of the firm itself. Therefore, SMEs can, with appropriate planning and the integration of marketing and environmental strategies, become, selectively, global firms, with better credit access and sales growth, concomitant with lower risks. The High Tech industrial SME will succeed better if, from the outset, they behave like the organization they want to become, with globalization a key element of strategy, from the business plan foreword. Two case studies are presented, one documenting the water sector and die other the cost savings possible by integrating environmental management into die firm’s strategy.     

Scoring functions in protein folding and design

We present an analysis of the assumptions behind some of the most commonly used methods for evaluating the goodness of the fit between a sequence and a structure. Our studies on a lattice model show that methods based on statistical considerations are easy to use and can capture some of the features of protein-like sequences and their corresponding native states, but unfortunately are incapable of recognizing, with certainty, the native-like conformation of a sequence among a set of decoys. Meanwhile, an optimization method, entailing the determination of the parameters of an effective free energy of interaction, is much more reliable in recognizing the native state of a sequence. However, the statistical method is shown to perform quite well in tests of protein design.     

Specific Expansion of Protein Families in the Radioresistant Bacterium Deinococcus Radiodurans

Computer analysis of the complete genome of Deinococcus radioduransR1 reveals a number of protein families, which are over-represented in this organism, compared to most other bacteria with known genome sequences. These families include both previously characterized and uncharacterized proteins. Most of the families whose functions are known or could be predicted seem to be related to stress-response and elimination of damage products (cell-cleaning). The two most prominent family expansions are the Nudix (MutT) family of pyrophosphohydrolases and a previously unnoticed family of proteins related to Bacillus subtilisDinB that could possess a metal-dependent enzymatic activity whose exact nature remains to be determined. Several proteins of the expanded families, particularly the Nudix family, are fused to other domains and form multidomain proteins that are so far unique for Deinococcus. The domain composition of some of these proteins indicates that they could be involved in novel DNA-repair pathways. Such unique proteins are good targets for knock-out and gene expression studies, which are aimed to shed light on the unusual features of this interesting10.6pt bacterium.     

Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts

 When pumpkin (Cucurbita spp., cv. Ebisu Nankin) ascorbate oxidase cDNA was introduced into cultured cells of tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow No. 2) by Agrobacterium-mediated transformation, the transgenic cells expressed and secreted the recombinant pumpkin ascorbate oxidase into the culture medium. These transgenic cells showed no morphological difference from wild-type cells. However, in the presence of applied hormones protoplasts prepared from the transgenic cells elongated more rapidly than those of wild-type cells. We propose that ascorbate oxidase may play a key role in the regulation of cell expansion perhaps by controlling transport processes through the plasma membrane, but not by affecting the cell wall. Received: 28 October 1999 / Accepted: 18 January 2000     

Plant leaf area expansion and assimilate production in wheat (Triticum aestivum L.) growing under low phosphorus conditions

Under phosphorus deficiency reductions in plant leaf area have been attributed to both direct effects of P on the individual leaf expansion rate and to a reduced availability of assimilates for leaf growth. In this work we use experimental and simulation techniques to identify and quantify these processes in wheat plants growing under P-deficient conditions. In a glasshouse experiment we studied the effects of soil P addition (0–138 kg P₂O₅ ha^-1) on tillering, leaf emergence, leaf expansion, plant growth, and leaf photosynthesis of wheat plants (cv. INTA Oasis) that were not water stressed. Plants were grown in pots containing a P-deficient (3 mg P g^-1 soil) sandy soil. Sowing and pots were arranged to simulate a crop stand of 173 plants m^-2. Experimental results were integrated in a simulation model to study the relative importance of each process in determining the plant leaf area during vegetative stages of wheat. Phosphorus deficiency significantly reduced plant leaf area and dry weight production. Under P-deficient conditions the phyllochron (PHY) was increased up to a 32%, compared to that of high-P plants. In low-P plants the rate of individual leaf area expansion during the quasi-linear phase of leaf expansion (LER) was significantly reduced. The effect of P deficiency on LER was the main determinant of the final size of the individual leaves. In recently expanded leaves phosphorus deficiency reduced the photosynthesis rate per unit leaf area at high radiation (AMAX), up to 57%. Relative values of AMAX showed an hyperbolic relationship with leaf P% saturating at 0.27%. Relative values of the tillering rate showed an hyperbolic relationship with the shoot P% saturating at values above 0.38%. The value of LER was not related to the concentration of P in leaves or shoots. A morphogenetic model of leaf area development and growth was developed to quantify the effect of assimilate supply at canopy level on total leaf area expansion, and to study the sensitivity of different model variables to changes in model parameters. Simulation results indicated that under mild P stress conditions up to 80% of the observed reduction in plant leaf area was due to the effects of P deficiency on leaf emergence and tillering. Under extreme P-deficient conditions the simulation model failed to explain the experimental results indicating that other factors not taken into account by the model, i.e. direct effects of P on leaf expansion, must have been active. Possible mechanisms of action of the direct effects of P on individual leaf expansion are discussed in this work.     

The CURLY LEAF gene controls both division and elongation of cells during the expansion of the leaf blade in Arabidopsis thaliana

The CURLY LEAF (CLF ) gene in Arabidopsis thaliana (L.) Heynh. is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems To clarify the function of CLF in organ development, we characterized clf mutants using an anatomical and genetic approach. The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions. A decrease both in the extent of cell elongation and in the number of cells was evident in the clf mutant leaves, suggesting that the CLF gene might be involved in the division and elongation of cells during leaf morphogenesis. An analysis of the development of clf mutant leaves revealed that the period during which cell division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type. Two phases in the elongation of cells were also recognized from this analysis. From analysis of an angustifolia clf double mutant, we found that the two phases of elongation of leaf cells were regulated independently by each gene. Thus, the CLF gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia. Received: 19 February 1998 / Accepted: 24 March 1998