Evolution of primate α and θ defensins revealed by analysis of genomes

Defensins are endogenous peptides with cysteine-rich antimicrobial ability that contribute to host defence against bacterial, fungal and viral infections. There are three subfamilies of defensins in primates: α, β and θ-defensins. α-defensins are most present in neutrophils and Paneth cells; β-defensins are involved in protecting the skin and the mucous membranes of the respiratory, genitourinary and gastrointestinal tracts; and θ-defensins are physically distinguished as the only known fully-cyclic peptides of animal origin, which are first isolated from rhesus macaques. All three kinds of defensins have six conserved cysteines, three intramolecular disulfide bonds, a net positive charge, and β-sheet regions. α and θ-defensins are closely related, comparative amino acid sequences showed that the difference between them is that θ-defensins have an additional stop codon limits the initial defensin domain peptides to 12 residues. Humans, chimpanzees and gorillas do not produce θ-defensin peptides due to a premature stop codon present in the signal sequence of all θ-defensin pseudogenes. By using comprehensive computational searches, here we report the discovery of complete repertoires of the α and θ-defensin gene family in ten primate species. Consistent with previous studies, our phylogenetic analyses showed all primate θ-defensins evident formed one distinct clusters evolved from α-defensins. β-defensins are ancestors of both α and θ-defensins. Human has two copies of DEFA1 and DEFT1P, and two extra DEFA3 and DEFA10P genes compared with gorilla. As different primates inhabit in quite different ecological niches, the production of species-specific α and θ-defensins and these highly evolved θ-defensins in old world monkeys would presumably allow them to better respond to the specific microbial challenges that they face.     

Decoupling of differentiation between traits and their underlying genes in response to divergent selection

We dissected the relationship between genetic differentiation (Q(ST)) for a trait and its underlying genes (G(STq), differentiation for a quantitative locus) in an evolutionary context, with the aim of identifying the conditions in which these two measurements are decoupled. We used two parameters (θ(B) and θ(W)) scaling the contributions of inter- and intrapopulation allelic covariation between genes controlling the trait of interest. We monitored the changes in θ(B) and θ(W), Q(ST) and G(STq) over successive generations of divergent and stabilizing selection, in simulations for an outcrossing species with extensive gene flow. The dynamics of these parameters are characterized by two phases. Initially, during the earliest generations, differentiation of the trait increases very rapidly and the principal and immediate driver of Q(ST) is θ(B). During subsequent generations, G(STq) increases steadily and makes an equal contribution to Q(ST). These results show that selection first captures beneficial allelic associations at different loci at different populations, and then targets changes in allelic frequencies. The same patterns are observed when environmental change modifies divergent selection, as shown by the very rapid response of θ(B) to the changes of selection regimes. We compare our results with previous experimental findings and consider their relevance to the detection of molecular signatures of natural selection.     

Light microscope study of mixed helices in reconstituted Salmonella flagella

In this morphological study of bacterial flagella, single flagellar filaments in solutions were photographed by dark-field light microscopy to determine parameters to describe their intact shapes. First, I measured pitches of helices I, II and III assumed by copolymers of flagellins from Salmonella strains SJ25 and SJ814 (Asakura & Iino, 1972), and combined the data with electron microscopic data of the contour length per period of each filament to calculate the pitch angles of the three helices. (The pitch angle is the angle between a tangent to the filament and the helical axis.)Secondly, filaments which consisted of two blocks assuming different helices were prepared by two-step copolymerization of SJ25 and SJ814 flagellins and the configurations of these mixed-type filaments were examined. In filaments of any mixed type, the axes of the constituent blocks were oriented at the same angle, called the “block angle” Ψ. This angle was found to be approximated by Ψ = 180 ° ? ¦θ₁ ? θ₂¦, where θ₁ and θ₂ are the pitch angles of the mixed helices. On the basis of this finding, the morphology of mixed-type filaments is discussed.     

A four compartment linear mammillary model

Studies are made for a four compartment model in which the central compartment is connected reversibly to three other compartments and the elimination occurs from the central compartment only. Identification of different distribution rate constants is made, with concentrations of drug in the central compartment at different times of observation being known. The solution depends on an optimization method in which the different unknowns are reduced to single variable with the help of Archimedes spiral. Thus, the solution requires the global minimum of a functional of single variable. Results are compared with those obtained by the generalized least square method.     

Analysis of protein crystallographic structural data: analysis of the orientation of peptide planes

A new dihedral angle parameter θ′, defined by C_i?1|N_i…C_i|N_i+1, involving pairs of backbone atoms in two adjacent peptide planes is used in this paper to analyse the relative orientation of the peptide planes in a protein chain. The equiangle contours for this parameter on the ?+ψ in an approximately linear way. Thus, θ′ serves as a good single parameter representation for chainfolding characterization by bringing out the relative orientation of successive peptide planes. Unlike the earlier proposed θ which gives the gross folding features, θ′ can be applied for nonregular helical features. Its utility in the detection of bend regions in protein chains is compared with that of θ through theoretically calculated tables as well as examples from actual proteins.     

Impact of wastewater fouling on contact angle

Capillary dependent systems are highly influenced by surface fouling and may degrade as material surface properties change. In anticipation of a spacecraft microgravity fluids management system exposed to highly variable wetting conditions, the impact of urine wastewater fouling on capillary contact angle was examined. The results indicate that, in general, surface fouling can decrease the contact angle when crystalline structures or biofilms form. Small crystalline growth on the order of 10 μm can lower advancing contact angles θ_adv by approximately 30°, while biofilm growth can lower it by approximately 15°. Vacuum drying of fouled surfaces increased θ_adv by about 8°, and defects greater in height than 5% of the capillary length increased θ_adv by approximately 30°. These trends may indicate that promotion of wastewater fouling may improve the performance of capillary dependent fluids management systems. These results may also influence terrestrial technologies, including medical catheters and sustainable wastewater treatment systems exposed to wastewater fouling.     

On the asymptotic behaviour of the pseudolikelihood ratio test statistic with boundary problems

This paper considers the asymptotic distribution of the likelihood ratio statistic T for testing a subset of parameter of interest θ, θ = (γ, η), H(0) : γ = γ(0), based on the pseudolikelihood L(θ, ??), where ?? is a consistent estimator of ?, the nuisance parameter. We show that the asymptotic distribution of T under H(0) is a weighted sum of independent chi-squared variables. Some sufficient conditions are provided for the limiting distribution to be a chi-squared variable. When the true value of the parameter of interest, θ(0), or the true value of the nuisance parameter, ?(0), lies on the boundary of parameter space, the problem is shown to be asymptotically equivalent to the problem of testing the restricted mean of a multivariate normal distribution based on one observation from a multivariate normal distribution with misspecified covariance matrix, or from a mixture of multivariate normal distributions. A variety of examples are provided for which the limiting distributions of T may be mixtures of chi-squared variables. We conducted simulation studies to examine the performance of the likelihood ratio test statistics in variance component models and teratological experiments.     

Possible Generational Effects of Habitat Degradation on Alligator Reproduction

ABSTRACT Population decline of the American alligator (Alligator mississippiensis) was observed in Lake Apopka in central Florida, USA, in the early 1980s. This decline was thought to result from adult mortality and nest failure caused by anthropogenic increases in sediment loads, nutrients, and contaminants. Reproductive impairment also was reported. Extensive restoration of marshes associated with Lake Apopka has been conducted, as well as some limited restoration measures on the lake. Monitoring by the Florida Fish and Wildlife Conservation Commission (FFWCC) has indicated that the adult alligator population began increasing in the early 1990s. We expected that the previously reported high proportion of complete nest failure (θ₀) during the 1980s may have decreased. We collected clutches from alligator nests in Lake Apopka from 1983 to 2003 and from 5 reference areas from 1988 to 1991, and we artificially incubated them. We used a Bayesian framework with Gibbs sampler of Markov chain Monte Carlo simulation to analyze θ₀. Estimated θ₀ was consistently higher in Lake Apopka compared with reference areas, and the difference in θ₀ ranged from 0.19 to 0.56. We conducted change point analysis to identify and test the significance of the change point in θ₀ in Lake Apopka between 1983 and 2003, indicating the point of reproductive recovery. The estimated Bayes factor strongly supported the single change point hypothesis against the no change point hypothesis. The major downward shift in θ₀ probably occurred in the mid-1990s, approximately a generation after the major population decline in the 1980s. Furthermore, estimated θ₀ values after the change point (0.21) were comparable with those of reference areas (0.07–0.31). These results combined with the monitoring by FFWCC seem to suggest that anthropogenic habitat degradation caused reproductive impairment of adult females and decreases in θ₀ occurred with the sexual maturity of a new generation of breeding females. Long-term monitoring is essential to understand population changes due to habitat restoration. Such information can be used as an input in planning and evaluating restoration activities.     

Bayes Estimator of Survival Probability from Randomly Censored Observations with Weibull Distribution of Time to Death

This paper deals with Bayes estimation of survival probability when the data are randomly censored. Such a situation arises in case of a clinical trial which extends for a limited period T. A fixed number of patients (n) are observed whose times to death have identical Weibull distribution with parameters β and θ. The maximum times of observation for different patients are also independent uniform variables as the patients arrive randomly throughout the trial. For the joint prior distribution of (β, θ) as suggested by Sinha and Kale (1980, page 137) Bayes estimator of survival probability at time t (0<t<T) has been obtained. Considering squared error loss function it is the mean of the survival probability with respect to the posterior distribution of (β, θ). This estimator is then compared with the maximum likelihood estimator, by simulation, for various values of β, θ and censoring percentage. The proposed estimator is found to be better under certain conditions.     

Variations in the expression of theta-C3H alloantigen on thymic and peripheral lymphocytes of newborn and young adult mice

The relative frequency of lymphocytes of mice showing varying degrees of surface θ-positivity (circumferential fluorescence) was recorded. Thymocytes were nearly 100% θ-positive. The relative proportions of θ-positive cells in Peyer's patches and lymph nodes of newborn mice varied in an almost identical fashion as a function of age. At 4 weeks of age and beyond, the relative numbers of θ-positive cells in Peyer's patches were consistently lower than in lymph nodes. As opposed to the predominance of thymocytes with complete rings, peripheralized thymic (T) lymphocytes showed a broad, age- and organ-dependent range of surface θ-positivity. These results suggest that surface θ may be lost rather rapidly upon emigration of lymphocytes from the thymus and/or that many θ-positive T cells with complete rings disappear within a short time. Variations in the relative proportion of complete rings on mesenteric lymph node cells on Days 1 and 4, were tentatively related to antigen-induced changes in the magnitude of thymocyte emigration.The pattern of surface θ-antigen of a given thymocyte or T cell with its size and DNA synthetic activity was compared. The findings suggest that incomplete ring fluorescence may especially be observed on proliferating lymphoblasts in the outer thymic cortex on their way to acquire the full complement of θ-antigen, and on medullary thymic lymphocytes or T cells having reentered mitotic activity, in response to antigenic and/or other microenvironmental stimuli. Our study yielded data consistent with the hypothesis that the progressive loss of surface θ-antigen does not represent a fully autonomous time-dependent process. Moreover, it is not clear if continued loss of θ-antigen by T cells below a certain threshold would render these cells undetectable by anti-θ sera.     

Characterization and biological treatment of pickling industry wastewater

Pickling industry wastewaters present unique difficulties in biological treatment because of high salt content (3–6% salt). Conventional activated sludge cultures disintegrate or loose microbial activity as a result of plasmolysis at salt concentrations above 1%. In order to overcome adverse effects of salt in pickling wastewater, salt tolerant bacteria (Halobacter halobium) was added to activated sludge culture and used in biological treatment of the wastewater in an activated sludge unit. After characterization and nutrient balancing of the wastewater, an activated sludge unit was used in laboratory to investigate the effects of major process variables such as sludge age and hydraulic residence time on performance of the system. Single stage and two stage activated processes were used for the treatment of the pickling wastewater. More than 95% of COD removal was obtained with a single stage process at a sludge age of θ_c=10?d and hydraulic residence time of θ_H=30?h. Similar results were obtained with the two stage process, when sludge ages and hydraulic residence times for each stage were θ_c1=θ_c2=10?d, and θ_H1=θ_H2=15?h, respectively. Kinetic coefficients were determined and the design equations were developed by using the experimental data.     

Polarization angle dependence of stark absorption spectra of spirilloxanthin bound to the reconstituted LH1 complexes using LH1-subunits isolated from the purple photosynthetic bacterium Rhodospirillum rubrum

Reconstituted LH1 complexes were prepared using the LH1 subunit-type complexes, isolated from the purple photosynthetic bacterium Rhodospirillum (Rs.) rubrum, and purified all-trans spirilloxanthin. Stark absorption spectra of spirilloxanthin bound to both the native and reconstituted LH1 complexes were compared in different polarization angles (χ) against the external electric field. From the polarization angle dependence of the Stark absorption spectra, two angles were determined in reference to the direction of transition dipole moment (m) of spirilloxanthin: one is the change in polarizability upon photoexcitation (Δα), θ(Δα) and the other is the change in static dipole moment upon photoexcitation (Δμ), θ(Δμ). Despite the symmetric molecular structure of all-trans spirilloxanthin, its Stark absorption spectra show pronounced values of Δμ. This large Δμ values essentially caused by the effect of induced dipole moment through Δα both in the cases for native and reconstituted LH1 complexes. However, slightly different values of θ(Δα) and θ(Δμ) observed for the native LH1 complex suggest that spirilloxanthin is asymmetrically distorted when bound to the native LH1 complex and gives rise to intrinsic Δμ value.     

Unconditional Non-Asymptotic One-Sided Tests for Independent Binomial Proportions When the Interest Lies in Showing Non-Inferiority and/or Superiority

For two independent binomial proportions Barnard (1947) has introduced a method to construct a non-asymptotic unconditional test by maximisation of the probabilities over the ‘classical’ null hypothesis H₀= {(θ₁, θ₂) ∈ [0, 1]²: θ₁ = θ₂}. It is shown that this method is also useful when studying test problems for different null hypotheses such as, for example, shifted null hypotheses of the form H₀ = {(θ₁, θ₂) ∈ [0, 1]²: θ₂ ≤ θ₁ ± Δ } for non-inferiority and 1-sided superiority problems (including the classical null hypothesis with a 1-sided alternative hypothesis). We will derive some results for the more general ‘shifted’ null hypotheses of the form H₀ = {(θ₁, θ₂) ∈ [0, 1]²: θ₂ ≤ g(θ₁ )} where g is a non decreasing curvilinear function of θ₁. Two examples for such null hypotheses in the regulatory setting are given. It is shown that the usual asymptotic approximations by the normal distribution may be quite unreliable. Non-asymptotic unconditional tests (and the corresponding p-values) may, therefore, be an alternative, particularly because the effort to compute non-asymptotic unconditional p-values for such more complex situations does not increase as compared to the classical situation. For ‘classical’ null hypotheses it is known that the number of possible p-values derived by the unconditional method is very large, albeit finite, and the same is true for the null hypotheses studied in this paper. In most of the situations investigated it becomes obvious that Barnard's CSM test (1947) when adapted to the respective null space is again a very powerful test. A theorem is provided which in addition to allowing fast algorithms to compute unconditional non-asymptotical p-values fills a methodological gap in the calculation of exact unconditional p-values as it is implemented, for example, in Stat Xact 3 for Windows (1995).     

A quantitative analysis of excluded-site effects for highly cooperative binding systems

Binding isotherms can provide quantitive information regarding the stability of a molecular complex. Theorectical studies in recent years have been directed to systems in which a single ligand can exclude more than one polymer site (excluded-site effect). This system has minium of thre parameters to describe the binding data: the intrinsic binding constant, B; the remote-neighbor cooperative paramaters, σ_q and the number of excluded sites, q. It is suggested in the present communication that precise values for these three parameters can be obtained by utilizing the characteristics of two forms of data representation: θ vs ln m and θ/m vs θ, where θ is the degree of saturation (0?θ?1) and m is the molality of free ligand. The matrix generation method is used to obtain empirical equations relating the midpoint location and slope at the midpoint of the θ vs ln m plot to the three molecular parameters. A modified Scatchard theory is also presented for highly cooperative systems, which results in an expression relaing the maximum in the θ/m vs θ plot to the molecular parameters σ_q and q, thus providing the third equation for the three unknown parameters. The novel method f analysis is illustrated with the AMP-poly(L -arginine) and oligocytidylate–T7 DNA sstems.     

Proportional Increase in the θ Carrying Lymphocytes in Peripheral Lymphoid Tissue following Treatment with Cyclophosphamide

WE have shown¹ that cyclophosphamide (CY) in a single dose of 300 mg/kg body weight selectively depletes those areas of mouse lymph nodes and spleen containing lymphocytes which are not dependent on thymus integrity in late foetal or early neonatal life. Depletion of similar areas was also produced by the same treatment in guinea-pigs. It was felt that this selective depletion could be demonstrated quantitatively by the use of the θ-antigen as a marker for T-lymphocytes² in the mouse.     

Species‐specific stomatal response of trees to drought – a link to vegetation dynamics?

Question: Is stomatal regulation specific for climate and tree species, and does it reveal species‐specific responses to drought? Is there a link to vegetation dynamics? Location: Dry inner alpine valley, Switzerland Methods: Stomatal aperture (θ_E) of Pinus sylvestris, Quercus pubescens, Juniperus communis and Picea abies were continuously estimated by the ratio of measured branch sap flow rates to potential transpiration rates (adapted Penman‐Monteith single leaf approach) at 10‐min intervals over four seasons. Results: θ_E proved to be specific for climate and species and revealed distinctly different drought responses: Pinus stomata close disproportionately more than neighbouring species under dry conditions, but has a higher θ_E than the other species when weather was relatively wet and cool. Quercus keeps stomata more open under drought stress but has a lower θ_E under humid conditions. Juniperus was most drought‐tolerant, whereas Picea stomata close almost completely during summer. Conclusions: The distinct microclimatic preferences of the four tree species in terms of θ_E strongly suggest that climate (change) is altering tree physiological performances and thus species‐specific competitiveness. Picea and Pinus currently live at the physiological limit of their ability to withstand increasing temperature and drought intensities at the sites investigated, whereas Quercus and Juniperus perform distinctly better. This corresponds, at least partially, with regional vegetation dynamics: Pinus has strongly declined, whereas Quercus has significantly increased in abundance in the past 30 years. We conclude that θ_E provides an indication of a species' ability to cope with current and predicted climate.     

Purification and Some Properties of Tetanolysin

Tetanolysin was purified from the culture fluid of a strain of Clostridium tetani by ammonium sulfate fractionation, acetone precipitation and repeated gel filtration. Two hemolysins with different molecular weights were separated by gel filtration, and the smaller one, tetanolysin, was further purified. The purification raised the specific activity of tetanolysin 1,050-fold to 500 HU/μg of protein. The purified preparation gave a single, relatively broad band on polyacrylamide gel electrophoresis, in which the activity was roughly parallel with the protein concentration. However, on sodium dodecylsulfate-gel electrophoresis it gave two bands with nearly equal amounts of proteins, showing molecular weights of 53,000 and 48,000±3,000. Furthermore, isoelectric focusing revealed four peaks of the activity whose isoelectric pHs were 6.1, 5.6, 5.3, and 6.6 in decreasing order of the activity. These findings suggest that the preparation contains four hemolysins with different pis, which are classifiable into two groups by molecular size. The preparation was completely free of tetanus neurotoxin and proteases. Tetanolysin was more strongly inhibited by cholesterol and more rapidly adsorbed onto erythrocytes than θ-toxin of Cl. perfringens.     

Evidence for linkage between K88ab, K88ac intestinal receptors to Escherichia coli and transferrin loci in pigs

Segregation at the loci coding for the K88ab and K88ac small intestinal receptors to E. coli adhesins (K88abR, K88acR) and at the transferrin (TF) locus was studied in 38 pig families including 273 piglets. The TF locus showed a segregation deviation towards the B variant while each of the K88 receptors behaved as a single autosomal dominant gene. Recombinants between K88abR and K88acR provide evidence that they are under the control of two different loci. Thirty-two triple backcross families were selected to test linkage and estimate recombination rates (θ). Our results demonstrate that the two K88 receptor loci are closely linked (θ= 0.02) with a maximum lod score value (Z_m) of 46.0. In addition, they are linked to the TF locus, θ= 0.14, Z_m= 19.6 for the K88abR locus and θ= 0.16, Z_m= 17.9 for the K88acR locus. The estimated recombination rates, smaller in males than in females, are consistent with the order TF-K88abR-K88acR. This linkage thus localizes the K88 loci, as the TF locus, on chromosome 13.     

θ-Defensins: Cyclic Peptides with Endless Potential

θ-Defensins, the only cyclic peptides of animal origin, have been isolated from the leukocytes of rhesus macaques and baboons. Their biogenesis is unusual because each peptide is an 18-residue chimera formed by the head-to-tail splicing of nonapeptides derived from two separate precursors. θ-Defensins have multiple arginines and a ladder-like tridisulfide array spanning their two antiparallel β-strands. Human θ-defensin genes contain a premature stop codon that prevents effective translation of the needed precursors; consequently, these peptides are not present in human leukocytes. Synthetic θ-defensins with sequences that correspond to those encoded within the human pseudogenes are called retrocyclins. Retrocyclin-1 inhibits the cellular entry of HIV-1, HSV, and influenza A virus. The rhesus θ-defensin RTD-1 protects mice from an experimental severe acute respiratory syndrome coronavirus infection, and retrocyclin-1 protects mice from infection by Bacillus anthracis spores. The small size, unique structure, and multiple host defense activities of θ-defensins make them intriguing potential therapeutic agents.