Estimating Finite Rate of Population Increase for Sharks Based on Vital Parameters

The vital parameter data for 62 stocks, covering 38 species, collected from the literature, including parameters of age, growth, and reproduction, were log-transformed and analyzed using multivariate analyses. Three groups were identified and empirical equations were developed for each to describe the relationships between the predicted finite rates of population increase (λ’) and the vital parameters, maximum age (T_max), age at maturity (T_m), annual fecundity (f/R_c)), size at birth (L_b), size at maturity (L_m), and asymptotic length (L_∞). Group (1) included species with slow growth rates (0.034 yr^-1 < k < 0.103 yr^-1) and extended longevity (26 yr < T_max < 81 yr), e.g., shortfin mako Isurus oxyrinchus, dusky shark Carcharhinus obscurus, etc.; Group (2) included species with fast growth rates (0.103 yr^-1 < k < 0.358 yr^-1) and short longevity (9 yr < T_max < 26 yr), e.g., starspotted smoothhound Mustelus manazo, gray smoothhound M. californicus, etc.; Group (3) included late maturing species (L_m/L_∞ ≧ 0.75) with moderate longevity (T_max < 29 yr), e.g., pelagic thresher Alopias pelagicus, sevengill shark Notorynchus cepedianus. The empirical equation for all data pooled was also developed. The λ’ values estimated by these empirical equations showed good agreement with those calculated using conventional demographic analysis. The predictability was further validated by an independent data set of three species. The empirical equations developed in this study not only reduce the uncertainties in estimation but also account for the difference in life history among groups. This method therefore provides an efficient and effective approach to the implementation of precautionary shark management measures.     

Simple Uniaxial and Uniform Biaxial Deformation of Nearly Isotropic Incompressible Tissues

A method is developed for analyzing in a unified manner both uniaxial and uniform biaxial strain data obtained from nearly isotropic tissues. The formulation is a direct application of nonlinear elasticity theory pertaining to large deformations. The general relation between Eulerian stress (σ) and extension ratio (λ) in soft isotropic elastic bodies undergoing uniform deformation takes the simple form: σ = ((λ³ - 1)/λ) f(λ), where f(λ) must be determined for each material. The extension ratio may be either greater than 1.0 (uniaxial elongation), or lie between zero and 1.0 (uniform biaxial extension). Simple analytical functions for f(λ) are most readily found for each tissue by plotting all data as (λ³ - 1)/λσ vs. λ. Of those tissues investigated in this way (dog pericardium and pleura, and cat mesentery and dura), all but pleura could be adequately described by a parabola: 1/f(λ) = 1/k{[(λ_M - λ)(λ - λ_m)]/[λ_M - λ_m}. In these instances, three material constants per tissue (K, λ_M, λ_m) served to predict approximately the stresses attained during both small and large deformations, in strips and sheets alike. It was further found that the uniaxial strain asymptote (λ_M) was linearly related to the biaxial strain asymptote (Λ_M), thus effectively reducing the number of constants by one.     

Signatures of neutral evolution in exponentially growing tumors: A theoretical perspective

Recent work of Sottoriva, Graham, and collaborators have led to the controversial claim that exponentially growing tumors have a site frequency spectrum that follows the 1/f law consistent with neutral evolution. This conclusion has been criticized based on data quality issues, statistical considerations, and simulation results. Here, we use rigorous mathematical arguments to investigate the site frequency spectrum in the two-type model of clonal evolution. If the fitnesses of the two types are λ₀ < λ₁, then the site frequency spectrum is c/f^α where α = λ₀/λ₁. This is due to the advantageous mutations that produce the founders of the type 1 population. Mutations within the growing type 0 and type 1 populations follow the 1/f law. Our results show that, in contrast to published criticisms, neutral evolution in an exponentially growing tumor can be distinguished from the two-type model using the site frequency spectrum.     

Deoxyribonucleic Acid Replication in λ Bacteriophage Mutants

After ultraviolet light induction of Escherichia coli K-12 strain W3350(λ), several structural intermediate forms of phage deoxyribonucleic acid (DNA) are synthesized. The early defective lysogens of λ, sus O₈, sus P₃, and T₁₁, were found to synthesize none of the DNA structural intermediates. A lysogen believed to be defective in all known phage activities, λsus N₇, was found to be able to synthesize an early phage DNA intermediate. The lysogen λsus Q₂₁, defective in late phage functions, is able to synthesize the early phage DNA intermediate and a concatenated molecule of greater molecular weight than the mature λ DNA.     

Random Coil to Globular Thermal Response of a Protein (H3.1) with Three Knowledge-Based Coarse-Grained Potentials

The effect of temperature on the conformation of a histone (H3.1) is studied by a coarse-grained Monte Carlo simulation based on three knowledge-based contact potentials (MJ, BT, BFKV). Despite unique energy and mobility profiles of its residues, the histone H3.1 undergoes a systematic (possibly continuous) structural transition from a random coil to a globular conformation on reducing the temperature. The range over which such a systematic response in variation of the radius of gyration (R_g) with the temperature (T) occurs, however, depends on the potential, i.e. ΔT_MJ ≈ 0.013–0.020, ΔT_BT ≈ 0.018–0.026, and ΔT_BFKV ≈ 0.006–0.013 (in reduced unit). Unlike MJ and BT potentials, results from the BFKV potential show an anomaly where the magnitude of R_g decreases on raising the temperature in a range ΔT_A ≈ 0.015–0.018 before reaching its steady-state random coil configuration. Scaling of the structure factor, S(q) ∝ q^−1/ν, with the wave vector, q = 2π/λ, and the wavelength, λ, reveals a systematic change in the effective dimension (D_e∼1/ν) of the histone with all potentials (MJ, BT, BFKV): D_e∼3 in the globular structure with D_e∼2 for the random coil. Reproducibility of the general yet unique (monotonic) structural transition of the protein H3.1 with the temperature (in contrast to non-monotonic structural response of a similar but different protein H2AX) with three interaction sets shows that the knowledge-based contact potential is viable tool to investigate structural response of proteins. Caution should be exercise with the quantitative comparisons due to differences in transition regimes with these interactions.     

Thermal Characterization,Crystal Field Analysis and In-Band Pumped Laser Performance of Er Doped NaY(WO4)2 Disordered Laser Crystals

Undoped and Er-doped NaY(WO₄)₂ disordered single crystals have been grown by the Czochralski technique. The specific heat and thermal conductivity (κ) of these crystals have been characterized from T = 4 K to 700 K and 360 K, respectively. It is shown that κ exhibits anisotropy characteristic of single crystals as well as a κ(T) behavior observed in glasses, with a saturation mean free phonon path of 3.6 Å and 4.5 Å for propagation along a and c crystal axes, respectively. The relative energy positions and irreducible representations of Stark Er³⁺ levels up to ⁴G_7/2 multiplet have been determined by the combination of experimental low (<10 K) temperature optical absorption and photoluminescence measurements and simulations with a single-electron Hamiltonian including both free-ion and crystal field interactions. Absorption, emission and gain cross sections of the ⁴I_13/2↔⁴I_15/2 laser related transition have been determined at 77 K. The ⁴I_13/2 Er³⁺ lifetime (τ) was measured in the temperature range of 77–300 K, and was found to change from τ (77K) ≈ 4.5 ms to τ (300K) ≈ 3.5 ms. Laser operation is demonstrated at 77 K and 300 K by resonantly pumping the ⁴I_13/2 multiplet at λ≈1500 nm with a broadband (FWHM≈20 nm) diode laser source perfectly matching the 77 K crystal ⁴I_15/2 → ⁴I_13/2 absorption profile. At 77 K as much as 5.5 W of output power were obtained in π-polarized configuration with a slope efficiency versus absorbed pump power of 57%, the free running laser wavelength in air was λ≈1611 nm with the laser output bandwidth of 3.5 nm. The laser emission was tunable over 30.7 nm, from 1590.7 nm to 1621.4 nm, for the same π-polarized configuration.     

State Transitions in the Green Alga Scenedesmus Obliquus Probed by Time-Resolved Chlorophyll Fluorescence Spectroscopy and Global Data Analysis

Decay-associated fluorescence spectra of the green alga Scenedesmus obliquus have been measured by single-photon timing with picosecond resolution in various states of light adaptation. The data have been analyzed by applying a global data analysis procedure. The amplitudes of the decay-associated spectra allow a determination of the relative antenna sizes of the photosystems. We arrive at the following conclusions: (a) The fluorescence kinetics of algal cells with open PS II centers (F₀ level) have to be described by a sum of three exponential components. These decay components are attributed to photosystem (PS) I (τ ≈ 85 ps, λ_max^em ≈ 695-700 nm), open PS II α-centers (τ ≈ 300 ps, λ_max^em = 685 nm), and open PS II β-centers (τ ≈ 600 ps, λ_max^em = 685 nm). A fourth component of very low amplitude (τ ≈ 2.2-2.3 ns, λ_max^em = 685 nm) derives from dead chlorophyll. (b) At the F_max level of fluorescence there are also three decay components. They originate from PS I with properties identical to those at the F₀ level, from closed PS II α-centers (τ ≈ 2.2 ns, λ_max^em = 685 nm) and from closed PS β-centers (τ ≈ 1.2 ns, λ_max^em = 685 nm). (c) The major effect of light-induced state transitions on the fluorescence kinetics involves a change in the relative antenna size of α- and β-units brought about by the reversible migration of light-harvesting complexes between α-centers and β-centers. (d) A transition to state II does not measurably increase the direct absorption cross-section (antenna size) of PS I. Our data can be rationalized in terms of a model of the antenna organization that relates the effects of state transitions and light-harvesting complex phosphorylation with the concepts of PS II α,β-heterogeneity. We discuss why our results are in disagreement with those of a recent lifetime study of Chlorella by M. Hodges and I. Moya (1986, Biochim. Biophys. Acta., 849:193-202).     

Renal Water Molecular Diffusion Characteristics in Healthy Native Kidneys: Assessment with Diffusion Tensor MR Imaging

Background

To explore the characteristics of diffusion tensor imaging (DTI) and magnetic resonance (MR) imaging in healthy native kidneys.

Methods

Seventy-three patients without chronic kidney disease underwent DTI-MRI with spin echo-echo planar (SE-EPI) sequences accompanied by an array spatial sensitivity encoding technique (ASSET). Cortical and medullary mean, axial and radial diffusivity (MD, AD and RD), fractional anisotropy (FA) and primary, secondary and tertiary eigenvalues (λ₁, λ₂, λ₃) were analysed in both kidneys and in different genders.

Results

Cortical MD, λ₂, λ₃, and RD values were higher than corresponding medullary values. The cortical FA value was lower than the medullary FA value. Medullary λ₁ and RD values in the left kidney were lower than in the right kidney. Medullary λ₂, and λ₃ values in women were higher than those in men. Medullary FA values in women were lower than those in men. Medullary FA (r = 0.351, P = 0.002) and λ₁ (r = 0.277, P = 0.018) positively correlated with eGFR. Medullary FA (r = −0.25, P = 0.033) negatively correlated with age.

Conclusions

Renal water molecular diffusion differences exist in human kidneys and genders. Age and eGFR correlate with medullary FA and primary eigenvalue.     

Diffusion Tensor Magnetic Resonance Imaging of the Pancreas

Purpose

To develop a diffusion-tensor-imaging (DTI) protocol that is sensitive to the complex diffusion and perfusion properties of the healthy and malignant pancreas tissues.

Materials and Methods

Twenty-eight healthy volunteers and nine patients with pancreatic-ductal-adenocacinoma (PDAC), were scanned at 3T with T2-weighted and DTI sequences. Healthy volunteers were also scanned with multi-b diffusion-weighted-imaging (DWI), whereas a standard clinical protocol complemented the PDAC patients’ scans. Image processing at pixel resolution yielded parametric maps of three directional diffusion coefficients λ1, λ2, λ3, apparent diffusion coefficient (ADC), and fractional anisotropy (FA), as well as a λ1-vector map, and a main diffusion-direction map.

Results

DTI measurements of healthy pancreatic tissue at b-values 0,500 s/mm²yielded: λ1 = (2.65±0.35)×10⁻³, λ2 = (1.87±0.22)×10⁻³, λ3 = (1.20±0.18)×10⁻³, ADC = (1.91±0.22)×10⁻³ (all in mm²/s units) and FA = 0.38±0.06. Using b-values of 100,500 s/mm² led to a significant reduction in λ1, λ2, λ3 and ADC (p<.0001) and a significant increase (p<0.0001) in FA. The reduction in the diffusion coefficients suggested a contribution of a fast intra-voxel-incoherent-motion (IVIM) component at b≤100 s/mm², which was confirmed by the multi-b DWI results. In PDACs, λ1, λ2, λ3 and ADC in both 0,500 s/mm² and 100,500 s/mm² b-values sets, as well as the reduction in these diffusion coefficients between the two sets, were significantly lower in comparison to the distal normal pancreatic tissue, suggesting higher cellularity and diminution of the fast-IVIM component in the cancer tissue.

Conclusion

DTI using two reference b-values 0 and 100 s/mm² enabled characterization of the water diffusion and anisotropy of the healthy pancreas, taking into account a contribution of IVIM. The reduction in the diffusion coefficients of PDAC, as compared to normal pancreatic tissue, and the smaller change in these coefficients in PDAC when the reference b-value was modified from 0 to 100 s/mm², helped identifying the presence of malignancy.     

The Stochastic Theory of Cell Proliferation

A stochastic theory of cell kinetics has been developed based on a realistic model of cell proliferation. A characteristic transit time, _i, has been assigned to each of the four states (G₁, S, G₂, M) of the cell cycle. The actual transit time, t_i, for any cell is represented by a distribution around _i with a variance σ_i². Analytic and computer formulations have been used to describe the time development of such characteristics as age distribution, labeling experiments, and response to perturbations of the system by, for example, irradiation and temperature. The decay of synchrony is analyzed in detail and is shown to proceed as a damped wave. From the first few peaks of the synchrony decay one can obtain the distribution function for the cell cycle time. The later peaks decay exponentially with a characteristic decay constant, λ, which depends only on the average cell-cycle time, , and the associated variance. It is shown that the system, upon any sudden disturbance, approaches new “equilibrium” proliferation characteristics via damped periodic transients, the damping being characterized by λ. Thus, the response time of the system, /λ, is as basic a parameter of the system as the cell-cycle time.     

Subunit Composition Determines the Single Channel Kinetics of the Epithelial Sodium Channel

We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of α with either wild-type β or γ subunits and α with carboxy-terminal truncated β (β_T) or γ (γ_T) subunits in Xenopus laevis oocytes. αβ and αβ_T channels (9.6 and 8.7 pS, respectively, with 150 mM Li⁺) were found to be constitutively open. Only upon inclusion of 1 μM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (P _o) for αβ was 0.54 and for αβ_T was 0.50. In comparison, αγ and αγ_T channels exhibited different kinetics: αγ channels (6.7 pS in Li⁺) had either long open times with short closings, resulting in a high P _o (0.78), or short openings with long closed times, resulting in a low P _o (0.16). The mean P _o for all αγ channels was 0.48. αγ_T (6.6 pS in Li⁺) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean P _o of 0.6, similar to that of αγ. Inclusion of 0.1 μM amiloride in the pipette solution reduced the mean open time of αγ_T to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of αβ, αβ_T (1 μM amiloride), and αγ_T (0.1 μM amiloride) channels. αβ and αβ_T had similar blocking and unblocking rate constants, whereas the unblocking rate constant for αγ_T was 10-fold slower than αβ_T. Our results indicate that subunit composition of ENaC is a main determinant of P _o. In addition, channel kinetics and P _o are not altered by carboxy-terminal deletion in the β subunit, whereas a similar deletion in the γ subunit affects channel kinetics but not P _o.     

Amide Proton Spin-Lattice Relaxation in Polypeptides: A Field-Dependence Study of the Proton and Nitrogen Dipolar Interactions in Alumichrome

The proton nuclear magnetic resonance (NMR) spin-lattice relaxation of all six amides of deferriferrichrome and of various alumichromes dissolved in hexadeutero-dimethylsulfoxide have been investigated at 100, 220, and 360 MHz. We find that, depending on the type of residue (glycyl or ornithyl), the amide proton relaxation rates are rather uniform in the metal-free cyclohexapeptide. In contrast, the ¹H spinlattice relaxation times (T₁'s) are distinct in the Al³⁺-coordination derivative. Similar patterns are observed in a number of isomorphic alumichrome homologues that differ in single-site residue substitutions, indicating that the spin-lattice relaxation rate is mainly determined by dipole-dipole interactions within a rigid molecular framework rather than by the specific primary structures. Analysis of the data in terms of ¹H—¹H distances (r) calculated from X-ray coordinates yields a satisfactory linear fit between T₁^-1 and Σr^-6 at the three magnetic fields. Considering the very sensitive r-dependence of T₁, the agreement gives confidence, at a quantitative level, both on the fitness of the crystallographic model to represent the alumichromes' solution conformation and on the validity of assuming isotropic rotational motion for the globular metallopeptides. An extra contribution to the amide proton T₁^-1 is proposed to mainly originate from the ¹H-¹⁴N dipolar interaction: this was supported by comparison with measurements on an ¹⁵N-enriched peptide. The nitrogen dipolar contribution to the peptide proton relaxation is discussed in the context of {¹H}—¹H nuclear Overhauser enhancement (NOE) studies because, especially at high fields, it can be dominant in determining the amide proton relaxation rates and hence result in a decreased effectiveness for the ¹H—¹H dipolar mechanism to cause NOE's. From the slope and intersect values of T₁^-1 vs. Σr^-6 linear plots, a number of independent estimates of τ_r, the rotational correlation time, were derived. These and the field-dependence of the T₁'s yield a best estimate <τ_r> ≈ 0.37 ns, in good agreement with 0.38 ns [unk] <τ_r> [unk] 0.41 ns, previously determined from ¹³C and ¹⁵N spin-lattice relaxation data.     

Mutations That Extend the Specificity of the Endonuclease Activity of λ Terminase

Terminase, an enzyme encoded by the Nu1 and A genes of bacteriophage lambda, is crucial for packaging concatemeric DNA into virions. cosN, a 22-bp segment, is the site on the virus chromosome where terminase introduces staggered nicks to cut the concatemer to generate unit-length virion chromosomes. Although cosN is rotationally symmetric, mutations in cosN have asymmetric effects. The cosN G₂C mutation (a G-to-C change at position 2) in the left half of cosN reduces the phage yield 10-fold, whereas the symmetric mutation cosN C₁₁G, in the right half of cosN, does not affect the burst size. The reduction in phage yield caused by cosN G₂C is correlated with a defect in cos cleavage. Three suppressors of the cosN G₂C mutation, A-E₅₁₅G, A-N₅₀₉K, and A-R₅₀₄C, have been isolated that restore the yield of λ cosN G₂C to the wild-type level. The suppressors are missense mutations that alter amino acids located near an ATPase domain of gpA. λ A-E₅₁₅G, A-N₅₀₉K, and A-R₅₀₄C phages, which are cosN⁺, also had wild-type burst sizes. In vitro cos cleavage experiments on cosN G₂C C₁₁G DNA showed that the rate of cleavage for A-E₅₁₅G terminase is three- to fourfold higher than for wild-type terminase. The A-E₅₁₅G mutation changes residue 515 of gpA from glutamic acid to glycine. Uncharged polar and hydrophobic residues at position 515 suppressed the growth defect of λ cosN G₂C C₁₁G. In contrast, basic (K, R) and acidic (E, D) residues at position 515 failed to suppress the growth defect of λ cosN G₂C C₁₁G. In a λ cosN⁺ background, all amino acids tested at position 515 were functional. These results suggest that A-E₅₁₅G plays an indirect role in extending the specificity of the endonuclease activity of λ terminase.     

Dissipation of the Membrane Potential in Susceptible Corn Mitochondria by the Toxin of Helminthosporium maydis, Race T, and Toxin Analogs

We have tested directly the effect of Helminthosporium maydis T (Hmt) toxin and various analogs on the membrane potential formed in mitochondria isolated from a Texas (T) cytoplasmic male-sterile and a normal (N) corn. ATP, malate or succinate generated a membrane potential (negative inside) as monitored by the absorbance change of a cationic dye, safranine. The relative membrane potential (Δψ) could also be detected indirectly as ⁴⁵Ca²⁺ uptake. Hmt toxin added to T mitochondria dissipated the steady state Δψ similar to addition of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (Cpd XIII: C₄₁H₆₈O₁₂ and Cpd IV: C₂₅H₄₄O₆), reduced native toxin (RT2C: C₄₁H₈₄O₁₃) and Pm toxin (band A: C₃₃H₆₀O₈, produced by the fungus, Phyllosticta maydis) were effective in dissipating Δψ and decreasing Ca²⁺ uptake with the following order: Pm (100) » HmT (23-30) > Cpd XIII (11-25) » RT2C (0-4−1.8) > Cpd IV (0.2−1.0). In contrast, the toxins and analogs had no effect on Δψ formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C₄₁H₆₈O₁₃) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the Δψ in energized mitochondria is caused mainly by the electrogenic extrusion of H⁺, the results support the idea that HmT toxin increases membrane permeability of T mitochondria to H⁺. The host specificity of the toxin suggests that an interaction with unique target site(s) on the inner mitochondrial membrane of T corn causes H⁺ leakage.     

Y chromosome lineage- and village-specific genes on chromosomes 1p22 and 6q27 control visceral leishmaniasis in Sudan

Familial clustering and ethnic differences suggest that visceral leishmaniasis caused by Leishmania donovani is under genetic control. A recent genome scan provided evidence for a major susceptibility gene on Chromosome 22q12 in the Aringa ethnic group in Sudan. We now report a genome-wide scan using 69 families with 173 affected relatives from two villages occupied by the related Masalit ethnic group. A primary ten-centimorgan scan followed by refined mapping provided evidence for major loci at 1p22 (LOD score 5.65; nominal p = 1.72 × 10⁻⁷; empirical p < 1 × 10⁻⁵; λ_S = 5.1) and 6q27 (LOD score 3.74; nominal p = 1.68 × 10⁻⁵; empirical p < 1 × 10⁻⁴; λ_S = 2.3) that were Y chromosome–lineage and village-specific. Neither village supported a visceral leishmaniasis susceptibility gene on 22q12. The results suggest strong lineage-specific genes due to founder effect and consanguinity in these recently immigrant populations. These chance events in ethnically uniform African populations provide a powerful resource in the search for genes and mechanisms that regulate this complex disease.     

Characterization of the 2',3' cyclic phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase-phosphatase and bacteriophage lambda phosphatase

Clostridium thermocellum polynucleotide kinase-phosphatase (CthPnkp) catalyzes 5′ and 3′ end-healing reactions that prepare broken RNA termini for sealing by RNA ligase. The central phosphatase domain of CthPnkp belongs to the dinuclear metallophosphoesterase superfamily exemplified by bacteriophage λ phosphatase (λ-Pase). CthPnkp is a Ni²⁺/Mn²⁺-dependent phosphodiesterase-monoesterase, active on nucleotide and non-nucleotide substrates, that can be transformed toward narrower metal and substrate specificities via mutations of the active site. Here we characterize the Mn²⁺-dependent 2′,3′ cyclic nucleotide phosphodiesterase activity of CthPnkp, the reaction most relevant to RNA repair pathways. We find that CthPnkp prefers a 2′,3′ cyclic phosphate to a 3′,5′ cyclic phosphate. A single H189D mutation imposes strict specificity for a 2′,3′ cyclic phosphate, which is cleaved to form a single 2′-NMP product. Analysis of the cyclic phosphodiesterase activities of mutated CthPnkp enzymes illuminates the active site and the structural features that affect substrate affinity and k_cat. We also characterize a previously unrecognized phosphodiesterase activity of λ-Pase, which catalyzes hydrolysis of bis-p-nitrophenyl phosphate. λ-Pase also has cyclic phosphodiesterase activity with nucleoside 2′,3′ cyclic phosphates, which it hydrolyzes to yield a mixture of 2′-NMP and 3′-NMP products. We discuss our results in light of available structural and functional data for other phosphodiesterase members of the binuclear metallophosphoesterase family and draw inferences about how differences in active site composition influence catalytic repertoire.     

Unique Plasmids Generated via pUC Replicon Mutagenesis in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

The plasmid pGKE75-cat_A138T, which comprises pUC18 and the cat_A138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CAT_A138T), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-cat_A138T in an error-prone thermophile, generating the mutant plasmid pGKE75^αβ-cat_A138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75^αβ-cat_A138T contained no mutation in the cat_A138T gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75^αβ-cat_A138T is attributable to increases in intracellular CAT_A138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75^αβ-cat_A138T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75^αβ-cat_A138T using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli.     

β-Lactam Effects on Mixed Cultures of Common Respiratory Isolates as an Approach to Treatment Effects on Nasopharyngeal Bacterial Population Dynamics

Background

Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae are bacteria present in the nasopharynx as part of normal flora. The ecological equilibrium in the nasopharynx can be disrupted by the presence of antibiotics.

Methodology/Principal Findings

A computerized two-compartment pharmacodynamic model was used to explore β-lactam effects on the evolution over time of a bacterial load containing common pharyngeal isolates by simulating free serum concentrations obtained with amoxicillin (AMX) 875 mg tid, amoxicillin/clavulanic acid (AMC) 875/125 mg tid and cefditoren (CDN) 400 mg bid regimens over 24 h. Strains and MICs (µg/ml) of AMX, AMC and CDN were: S. pyogenes (0.03, 0.03 and 0.015), S. pneumoniae (2, 2 and 0.25), a β-lactamase positive H. influenzae (BL⁺; >16, 2 and 0.06) and a β-lactamase positive AMC-resistant H. influenzae (BLPACR, >16, 8 and 0.06). Mixture of identical 1∶1∶1∶1 volumes of each bacterial suspension were prepared yielding an inocula of ≈4×10⁶ cfu/ml. Antibiotic concentrations were measured both in bacterial and in bacteria-free antibiotic simulations. β-lactamase production decreased AMX concentrations and fT_>MIC against S. pneumoniae (from 43.2% to 17.7%) or S. pyogenes (from 99.9% to 24.9%), and eradication was precluded. The presence of clavulanic acid countered this effect of co-pathogenicity, and S. pyogenes (but not BL⁺ and S. pneumoniae) was eradicated. Resistance of CDN to TEM β-lactamase avoided this co-pathogenicity effect, and CDN eradicated S. pyogenes and H. influenzae strains (fT_>MIC >58%), and reduced in 94% S. pneumoniae counts (fT_>MIC ≈25%).

Conclusions/Significance

Co-pathogenicity seems to be gradual since clavulanic acid countered this effect for strains very susceptible to AMX as S. pyogenes but not for strains with AMX MIC values in the limit of susceptibility as S. pneumoniae. There is a potential therapeutic advantage for β-lactamase resistant cephalosporins with high activity against streptococci.     

The influence of chloride and other univalent inorganic anions on the visible-absorption spectrum of aspartate aminotransferase

1. The influence of Cl⁻, Br⁻, NO₃⁻ and F⁻ ions on the visible-absorption spectrum of deionized aspartate aminotransferase was investigated. 2. Except for F⁻, these anions caused an increase of the extinction at 430mμ with a concomitant decrease of that at 362mμ. 3. The affinity constants for Cl⁻ and NO₃⁻ ions were calculated by a procedure based on the assumption that the anion stabilizes the protonated form of the enzyme chromophore (λ_max. 430mμ). 4. The true pK of the chromophore of the enzyme was found to be 5·25.