Divergent evolution of (betaalpha)8-barrel enzymes.

The (betaalpha)8-barrel is the most versatile and most frequently encountered fold among enzymes. It is an interesting question how the contemporary (betaalpha)8-barrels are evolutionarily related and by which mechanisms they evolved from more simple precursors. Comprehensive comparisons of amino acid sequences and three-dimensional structures suggest that a large fraction of the known (betaalpha)8-barrels have divergently evolved from a common ancestor. The mutational interconversion of enzymatic activities of several (betaalpha)8-barrels further supports their common evolutionary origin. Moreover, the high structural similarity between the N- and C-terminal (betaalpha)4 units of two (betaalpha)8-barrel enzymes from histidine biosynthesis indicates that the contemporary proteins evolved by tandem duplication and fusion of the gene of an ancestral 'half-barrel' precursor. In support of this hypothesis, recombinantly produced 'half-barrels' were shown to be folded, dimeric proteins.

Induction of antitumor immunity via intratumoral tetra-costimulator protein transfer.

Our group recently described a novel two-step Fc(gamma1) fusion protein transfer method, which entails the docking of Fc(gamma1) fusion proteins onto cells precoated with chemically palmitated protein A (pal-prot A). In the present study, we have adapted this protein transfer method, originally used in an ex vivo context, for in situ tumor cell engineering, and in so doing, we have evaluated its utility for the induction of antitumor immunity via combinatorial costimulator protein transfer on to tumor cell surfaces. The feasibility of "painting" cells with preformed conjugates of a murine B7-1 costimulator derivative, B7-1.Fc(gamma1), and pal-prot A in a single step was first established ex vivo. Next, B7-1.Fc(gamma1):pal-prot A transfer was accomplished in vivo by directly injecting the preformed conjugates into highly aggressive L5178Y-R lymphomas grown intradermally in syngeneic mice. The presence of cell surface-associated B7-1 epitopes on cells of the injected tumors was documented by flow cytometric analysis of cells recovered subsequently from the injected tumors. B7-1.Fc(gamma1), along with Fc(gamma1) fusion protein derivatives of three additional costimulators (Fc(gamma1).4-1BBL, CD48.Fc(gamma1), and Fc(gamma1).CD40L) geared toward a variety of immune effectors, were together preconjugated with pal-prot A and injected directly into tumor beds. Significantly, this "tetra-costimulator" combination, delivered intratumorally, induced complete tumor regression in approximately 45% of treated mice, whereas control injections of pal-prot A alone had no therapeutic effect. Furthermore, there was evidence for systemic antitumor immunity in that tumor-specific CTLs were detected in spleens recovered from cured mice, and these mice were uniformly protected against tumor rechallenge at distant tumor sites. Hence, combinatorial costimulator transfer, coupled to intratumoral delivery, may have special advantages for the induction of antitumor immunity.

Stability, catalytic versatility and evolution of the (beta alpha)(8)-barrel fold.

The (beta alpha)(8)-barrel is a versatile single-domain protein fold that is adopted by a large number of enzymes. The (beta alpha)(8)-barrel fold has been used as a model to elucidate the structural basis of protein thermostability and in studies to interconvert catalytic activities or substrate specificities by rational design or directed evolution. Recently, the (beta alpha)(4)-half-barrel was identified as a possible structural subdomain.

Imidazole glycerol phosphate synthase from Thermotoga maritima. Quaternary structure, steady-state kinetics, and reaction mechanism of the bienzyme complex.

Imidazole glycerol phosphate synthase, which links histidine and de novo purine biosynthesis, is a member of the glutamine amidotransferase family. In bacteria, imidazole glycerol phosphate synthase constitutes a bienzyme complex of the glutaminase subunit HisH and the synthase subunit HisF. Nascent ammonia produced by HisH reacts at the active site of HisF with N'-((5'-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield the products imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribotide. In order to elucidate the interactions between HisH and HisF and the catalytic mechanism of the HisF reaction, the enzymes tHisH and tHisF from Thermotoga maritima were produced in Escherichia coli, purified, and characterized. Isolated tHisH showed no detectable glutaminase activity but was stimulated by complex formation with tHisF to which either the product imidazole glycerol phosphate or a substrate analogue were bound. Eight conserved amino acids at the putative active site of tHisF were exchanged by site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics. Aspartate 11 appeared to be essential for the synthase activity both in vitro and in vivo, and aspartate 130 could be partially replaced only by glutamate. The carboxylate groups of these residues could provide general acid/base catalysis in the proposed catalytic mechanism of the synthase reaction.

Thermophilic adaptation of proteins.

Hyperthermophilic organisms optimally grow close to the boiling point of water. As a consequence, their macromolecules must be much more thermostable than those from mesophilic species. Here, proteins from hyperthermophiles and mesophiles are compared with respect to their thermodynamic and kinetic stabilities. The known differences in amino acid sequences and three-dimensional structures between intrinsically thermostable and thermolabile proteins will be summarized, and the crucial role of electrostatic interactions for protein stability at high temperatures will be highlighted. Successful attempts to increase the thermostability of proteins, which were either based on rational design or on directed evolution, are presented. The relationship between high thermo-stability of enzymes from hyperthermophiles and their low catalytic activity at room temperature is discussed. Not all proteins from hyperthermophiles are thermostable enough to retain their structures and functions at the high physiological temperatures. It will be shown how this shortcoming can be surpassed by extrinsic factors such as large molecular chaperones and small compatible solutes. Finally, the potential of thermostable enzymes for biotechnology is discussed.

Assessment of the reliability of high-intensity quadriceps femoris muscle fatigue.

OBJECTIVE: The purpose of this study was to evaluate test-retest reliability of two different measures of isokinetic quadriceps muscle fatigue. METHODS: Subjects for this study included 16 healthy, college age volunteers. Each subject performed 30 maximal, concentric repetitions on the Biodex System II Isokinetic Dynamometer at a preset angular velocity of 180 degrees.s(-1) for both the dominant and nondominant legs. Quadriceps work was evaluated between an angular range of 10 degrees and 60 degrees of flexion for each repetition. Quadriceps muscle fatigue was calculated through a fatigue index (work performed last 5 repetitions/work performed first five repetitions x 100) and the linear slope (beta) across the 30 repetitions. The subjects participated in two test sessions separated by one to two weeks. Intraclass correlation coefficients (ICC) and standard errors of measurements (SEM) were calculated for each fatigue measure on both legs. RESULTS: The findings demonstrated moderate to high ICCs for the nondominant leg (ICC = 0.78--0.92) and high ICCs for the slope and y-intercept for the dominant leg (ICC = 0.82 and 0.89, respectively). The fatigue index for the dominant leg was found to be low (ICC = 0.26). CONCLUSIONS: The findings of this study suggest that the quantification of muscle fatigue during high-intensity, short-term exercise is more reliably described by the slope, which is related to the magnitude of force output.

Dissection of a (betaalpha)8-barrel enzyme into two folded halves.

The (betaalpha)8-barrel, which is the most frequently encountered protein fold, is generally considered to consist of a single structural domain. However, the X-ray structure of the imidazoleglycerol phosphate synthase (HisF) from Thermotoga maritima has identified it as a (betaalpha) 8-barrel made up of two superimposable subdomains (HisF-N and HisF-C). HisF-N consists of the four N-terminal (betaalpha) units and HisF-C of the four C-terminal (betaalpha) units. It has been postulated, therefore, that HisF evolved by tandem duplication and fusion from an ancestral half-barrel. To test this hypothesis, HisF-N and HisF-C were produced in Escherichia coli, purified and characterized. Separately, HisF-N and HisF-C are folded proteins, but are catalytically inactive. Upon co-expression in vivo or joint refolding in vitro, HisF-N and HisF-C assemble to the stoichiometric and catalytically fully active HisF-NC complex. These findings support the hypothesis that the (betaalpha)8-barrel of HisF evolved from an ancestral half-barrel and have implications for the folding mechanism of the members of this large protein family.

Nutritional constraints in terrestrial and freshwater food webs.

Biological and environmental contrasts between aquatic and terrestrial systems have hindered analyses of community and ecosystem structure across Earth's diverse habitats. Ecological stoichiometry provides an integrative approach for such analyses, as all organisms are composed of the same major elements (C, N, P) whose balance affects production, nutrient cycling, and food-web dynamics. Here we show both similarities and differences in the C:N:P ratios of primary producers (autotrophs) and invertebrate primary consumers (herbivores) across habitats. Terrestrial food webs are built on an extremely nutrient-poor autotroph base with C:P and C:N ratios higher than in lake particulate matter, although the N:P ratios are nearly identical. Terrestrial herbivores (insects) and their freshwater counterparts (zooplankton) are nutrient-rich and indistinguishable in C:N:P stoichiometry. In both lakes and terrestrial systems, herbivores should have low growth efficiencies (10-30%) when consuming autotrophs with typical carbon-to-nutrient ratios. These stoichiometric constraints on herbivore growth appear to be qualitatively similar and widespread in both environments.

Structural evidence for evolution of the beta/alpha barrel scaffold by gene duplication and fusion.

The atomic structures of two proteins in the histidine biosynthesis pathway consist of beta/alpha barrels with a twofold repeat pattern. It is likely that these proteins evolved by twofold gene duplication and gene fusion from a common half-barrel ancestor. These ancestral domains are not visible as independent domains in the extant proteins but can be inferred from a combination of sequence and structural analysis. The detection of subdomain structures may be useful in efforts to search genome sequences for functionally and structurally related proteins.

Directed evolution of a (beta alpha)8-barrel enzyme to catalyze related reactions in two different metabolic pathways.

Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC ) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC ), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (betaalpha)(8)-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (betaalpha)(8)-barrel enzymes are very suitable for the design of new catalytic activities.

Strychnine alkaloid and avian reproduction: effects occur at lower dietary concentrations with mallard ducks than with bobwhite quail.

Separate subchronic reproductive toxicity studies were conducted using mallard ducks (Anas platyrhynchos) and northern bobwhite quail (Colinus virginianus). Three groups (32/group; 16 male-female pairs) of 17-week-old ducks (F0 generation) were fed Purina Game Bird Breeder Layena diets containing mean (+/-SD) 33.2 (+/-2.7), 68.9 (+/-1.8), and 140.9 (+/-5.1) microg/g strychnine for 20 weeks, with some pairs in each group fed control diet during a subsequent 3-week recovery period. Three groups (32/group; 16 male-female pairs) of 19-week-old quail (F0 generation) were fed similar diets containing mean (+/-SD) 279.2 (+/-10.1), 557.4 (+/-43.5), and 1,113.6 (+/-46.6) microg/g strychnine for 22 weeks without a recovery period. Separate groups of ducks and quail (32/group; 16 male-female pairs) were also fed control diets (0.0 microg/g strychnine) in each study. There were 16 weekly collections of eggs for the mallard study (13 for the diet-exposure period and 3 for the recovery period), and 11 collections for the quail study. Eggs laid during the last 13 and 10 weeks of the diet-exposure periods for ducks (plus 3 weeks of the recovery period) and quail, respectively, were incubated. Each hatch of F1 generation ducklings and chicks was observed for 14 days. Key results were: (1) the no observed adverse effect levels (NOAELs) for F0 ducks and quail were 33.2 and 1,113.6 microg/g strychnine, respectively--quail showed no reproductive effects at the current doses; (2) decreased egg production and hatching success occurred for mallard hens fed mean 140.9 microg/g strychnine diets; and (3) "normal-hatching" ducklings from eggs of F0 mallards fed mean 140.9 microg/g strychnine diets suffered greater mortality than ducklings from the other diet groups. Possible mechanisms of strychnine action on avian reproduction are discussed.

Structure and function of mutationally generated monomers of dimeric phosphoribosylanthranilate isomerase from Thermotoga maritima.

BACKGROUND: Oligomeric proteins may have been selected for in hyperthermophiles because subunit association provides extra stabilization. Phosphoribosylanthranilate isomerase (PRAI) is monomeric and labile in most mesophilic microorganisms, but dimeric and stable in the hyperthermophile Thermotoga maritima (tPRAI). The two subunits of tPRAI are associated back-to-back and are locked together by a hydrophobic loop. The hypothesis that dimerization is important for thermostability has been tested by rationally designing monomeric variants of tPRAI. RESULTS: The comparison of tPRAI and PRAI from Escherichia coli (ePRAI) suggested that levelling the nonplanar dimer interface would weaken the association. The deletion of two residues in the loop loosened the dimer. Subsequent filling of the adjacent pocket and the exchange of polar for apolar residues yielded a weakly associating and a nonassociating monomeric variant. Both variants are as active as the parental dimer but far more thermolabile. The thermostability of the weakly associating monomer increased significantly with increasing protein concentration. The X-ray structure of the nonassociating monomer differed from that of the parental subunit only in the restructured interface. The orientation of the original subunits was maintained in a crystal contact between two monomers. CONCLUSIONS: tPRAI is dimeric for reasons of stability. The clearly separated responsibilities of the betaalpha loops, which are involved in activity, and the alphabeta loops, which are involved in protein stability, has permitted the evolution of dimers without compromising their activity. The preserved interaction in the crystal contacts suggests the most likely model for dimer evolution.

Capsicum-laden soils decrease contact time by northern pocket gophers.

Fossorial rodents damage lawns/water impoundments/crops. We conducted a two-choice, parametric-type study to determine the effects of capsicum-oleoresin/soil mixtures (0.00, 0.75, 1.50, and 2.25%) upon soil-contact, soil-digging, and pelage-grooming behaviors in northern pocket gophers (Thomomys talpoides). In 3 alternate-day (1-h/day) exposures to > or = 1.50% capsicum-oleoresin soil mixtures, gophers decreased mean soil contact time by 46% relative to placebo-exposed animals. Grooming time yielded a concentration x trial interaction that showed intense grooming by capsicum-exposed animals during trial 1, with "convergence" of times to near those of the "placebos" (0.00% capsicum oleoresin) by trial 3. The significant decrease in grooming activity was attributed to the gophers' reduced contact with capsicum soil across repeated exposures, rather than to chemical habituation. Soil-digging behaviors were minimally affected. Results demonstrate the feasibility of deterring gopher habitation by mixing chemical irritants in soil.

Balance training for persons with functionally unstable ankles.

STUDY DESIGN: A nonrandomized 2-group pretest-posttest design. OBJECTIVES: To determine the effects of a 4-week balance training program during stance on a single leg. BACKGROUND: Individuals who have experienced multiple episodes of inversion ankle sprains often participate in balance training programs. Balance training is performed to treat existing proprioceptive deficits and to restore ankle joint stability, presumably by retraining altered afferent neuromuscular pathways. The effectiveness of such programs on individuals with functionally unstable ankles has yet to be established. METHODS AND MEASURES: Prior to and following training, subjects with self-reported functionally unstable ankles (5 women and 8 men, mean age = 21.9 +/- 3.1 years) and nonimpaired subjects (6 women and 7 men, mean age = 21.2 +/- 2.5 years) completed a static balance assessment for both limbs as well as the ankle joint functional assessment tool questionnaire (AJFAT). The subjects from both groups participated in a unilateral, multilevel, static and dynamic balance training program 3 times a week for 4 weeks. Subjects from the experimental group trained only the involved limb, and the nonimpaired group trained a randomly selected limb. A stability index (SI) was calculated during the balance assessment to indicate the amount of platform motion. Compared to low stability indices, high stability indices indicate greater platform motion during stance and therefore less stability. RESULTS: Following training, subjects from both groups demonstrated significant improvements in balance ability. When balance was assessed at a low resistance to platform tilt (stability level 2), the posttraining scores of both the subjects with unstable ankles (mean SI = 2.63 +/- 1.92) and the nonimpaired subjects (mean SI = 2.69 +/- 2.32) were significantly better than their pretraining scores (mean SIs = 5.93 +/- 3.65 and 4.67 +/- 3.43, respectively). Assessed at a high resistance to platform tilt (stability level 6), the posttraining scores of both subjects with unstable ankles (mean SI = 1.27 +/- 0.66) and the nonimpaired subjects (mean SI = 1.37 +/- 0.66) were significantly better than their pretraining scores (mean SIs = 2.30 +/- 1.88 and 2.04 +/- 1.43, respectively). Additionally, the posttraining AJFAT scores of subjects with unstable ankles (25.78 +/- 3.80) and the nonimpaired subjects (29.15 +/- 5.27) were significantly greater than their pretraining scores (17.11 +/- 3.44 and 22.92 +/- 5.22, respectively), indicating an overall improvement in perceived ankle joint functional stability. CONCLUSIONS: This study suggests that balance training is an effective means of improving joint proprioception and single-leg standing ability in subjects with unstable and nonimpaired ankles.

Efficient expression, purification and crystallisation of two hyperthermostable enzymes of histidine biosynthesis.

Enzymes from hyperthermophiles can be efficiently purified after expression in mesophilic hosts and are well-suited for crystallisation attempts. Two enzymes of histidine biosynthesis from Thermotoga maritima, N'-((5'-phosphoribosyl)-formimino)-5-aminoimidazol-4-carb oxamid ribonucleotide isomerase and the cyclase moiety of imidazoleglycerol phosphate synthase, were overexpressed in Escherichia coli, both in their native and seleno-methionine-labelled forms, purified by heat precipitation of host proteins and crystallised. N'-((5'-phosphoribosyl)-formimino)-5-aminoimidazol-4-carb oxamid ribonucleotide isomerase crystallised in four different forms, all suitable for X-ray structure solution, and the cyclase moiety of imidazoleglycerol phosphate synthase yielded one crystal form that diffracted to atomic resolution. The obtained crystals will enable the determination of the first three-dimensional structures of enzymes from the histidine biosynthetic pathway.

A histidine gene cluster of the hyperthermophile Thermotoga maritima: sequence analysis and evolutionary significance.

The sequences of histidine operon genes in hyperthermophiles are informative for understanding high protein thermostability and the evolution of metabolic pathways. Therefore, a cluster of eight his genes from the hyperthermophilic and phylogenetically early bacterium Thermotoga maritima was cloned and sequenced. The cluster has the gene order hisDCBdHAFI-E, lacking only hisG and hisBp, and does not contain intercistronic regions. This compact organization of his genes resembles the his operon of enterobacteria. Sequence analysis downstream of the stop codon of hisI-E identifies a region with a significantly higher cytosine over guanosine content, which is indicative of a rho-dependent termination of transcription of the his operon. Multiple sequence alignments of N1-((5'-phosphoribosyl)-formimino)-5-aminoimidazole-4-carboxyam ide ribonucleotide isomerase (HisA) and of the cycloligase moiety of imidazoleglycerol phosphate synthase (HisF) support the previous assignment of the (beta alpha)8-barrel fold to these proteins. The alignments also reveal a second phosphate-binding motif located in the first halves of both enzymes and thereby support the hypothesis that HisA and HisF have evolved by a sequence of two gene duplication events. Comparison of the amino acid compositions of HisA and HisF from mesophiles and thermophiles shows that the thermostable variants of both enzymes contain a significantly increased number of charged amino acid residues and may therefore be stabilized by additional salt bridges.

Subchronic dietary toxicity of strychnine: bobwhite quail are less sensitive than mallard ducks.

Separate, 28-day, subchronic studies of strychnine dietary toxicity were conducted using northern bobwhite quail (Colinus virginianus) and mallard ducks (Anas platyrhynchos). Five groups (five males five females/group) of 29-week-old quail were fed Purina(R) Game Bird Breeder Layena(R) diets containing mean (+/-SD) 484.2 (+/-17.0), 972. 6 (+/-54.0), 1,870.8 (+/-176.1), 3,516.7 (+/-68.0), and 6,083.3 (+/-269.6) microgram/g strychnine; whereas five groups of 27-week-old mallards (five males five females/group) were fed similar diets containing mean (+/-SD) 18.8 (+/-1.3), 91.1 (+/-27.3), 235.0 (+/-33. 8), 484.2 (+/-17.0), and 972.6 (+/-54.0) microgram/g strychnine. Separate "vehicle control" (0.0 microgram/g strychnine) groups (five males, five females/group) were included in each study. Strychnine toxicity was much less pronounced in quail; no observed effect concentrations (NOECs) were 972.6 (+/-54.0) and 91.1 (+/-27.3) microgram/g strychnine for quail and ducks, respectively. Several possible explanations for the species effects are offered, and some practical issues affecting the conduct of long-term, dietary toxicity studies are discussed.

The effects of muscular fatigue on shoulder proprioception.

OBJECTIVE: To assess the influence of muscular fatigue on active and passive shoulder proprioception within the midrange of rotation. DESIGN: A randomized controlled, before-and-after design. SETTING: Neuromuscular research laboratory. PARTICIPANTS: Twenty recreationally active men (mean age, 23.81+/-2.77 years) were randomly assigned to either a control or a fatigue group. Exclusion criteria were any history of upper extremity injury or pathology, cardiovascular disease, or disease affecting the sensory system. INTERVENTION: Shoulder proprioception was assessed by active reproduction of passive positioning (ARPP), active reproduction of active positioning (ARAP), reproduction of passive positioning (RPP), and threshold to detect passive motion (TTDPM). For each test direction, the experimental group performed two bouts of maximal reciprocal concentric isokinetic internal and external contractions at 180 degrees/s until peak torque decreased to 50% of the established maximum voluntary contraction. After two bouts of the fatigue protocol, subjects were randomly assessed for proprioception into internal or external rotation. MAIN OUTCOME MEASURES: The absolute angular error for active and passive proprioception was measured on the Biodex System II Isokinetic Dynamometer (Biodex Medical Inc., Shirley, NY, U.S.A.) and a proprioception testing device, respectively. MAIN RESULTS: A two-factor repeated measures analysis of variance revealed no significant interactions between the experimental and control groups for ARPP, ARAP, RPP, or TTDPM. CONCLUSIONS: Shoulder proprioception was not affected by the short-duration, high-intensity protocol used in this study. This may be due to the lack of an extended recovery period observed with this type of fatigue regimen.