On Nei and Roychoudhury''s Sampling Variances of Heterozygosity and Genetic Distance

Nei and Roychoudhury have developed an inequality relationship between the expected values of (j_x- g)² and (ĝ-g)² where j_x is a biased and ĝ is an unbiased estimate of population homozygosity g. Their inequality is somewhat weak and can be improved, as is demonstrated in this paper.     

Substrate specificity of the Bacillus licheniformis lyxose isomerase YdaE and its application in in vitro catalysis for bioproduction of lyxose and glucose by two-step isomerization

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on d-lyxose, suggesting that the enzyme is d-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²⁺. The apparent K_m values for d-lyxose and d-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k_cat/K_m) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for d-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of d-lyxose and d-glucose from d-xylose and d-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM d-lyxose and d-mannose, 3.7 mM and 3.8 mM d-lyxose and d-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.     

The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala⁴-α-d-Lys³ cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala¹(α→α)d-Lys³ and d-Ala⁴(α→α)d-Lys³ types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)² cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala¹-γ-d-Glu²-X³-d-Ala⁴-d-Ala⁵, where X is most often meso-diaminopimelic acid (meso-A₂pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala⁴-d-Ala⁵ bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala⁴ and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X³-d-Ala⁴ bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A₂pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link.     

Increase in Quantitative Variation After Exposure to Environmental Stresses and/or Introduction of a Major Mutation: G × E Interaction and Epistasis or Canalization?

Why does phenotypic variation increase upon exposure of the population to environmental stresses or introduction of a major mutation? It has usually been interpreted as evidence of canalization (or robustness) of the wild-type genotype; but an alternative population genetic theory has been suggested by J. Hermisson and G. Wagner: “the release of hidden genetic variation is a generic property of models with epistasis or genotype–environment interaction.” In this note we expand their model to include a pleiotropic fitness effect and a direct effect on residual variance of mutant alleles. We show that both the genetic and environmental variances increase after the genetic or environmental change, but these increases could be very limited if there is strong pleiotropic selection. On the basis of more realistic selection models, our analysis lends further support to the genetic theory of Hermisson and Wagner as an interpretation of hidden variance.A common experimental observation in quantitative genetics is a higher phenotypic variance for quantitative traits in populations that carry a major mutation or are exposed to environmental stresses (e.g., heat shock) (Scharloo 1991; for a recent review see Gibson and Dworkin 2004). Part of the added variance must be genetic because the population responds to artificial selection. The lower variability of the wild type than that of the mutants has been interpreted as evidence for robustness or canalization (Waddington 1957): that is, under the new condition the magnitudes of gene effects across all trait loci increase relative to the original condition. The importance of canalization has been recognized for a long time and has been the subject of renewed interest recently (see de Visser et al. 2003 and Hansen 2006 for reviews).An alternative population genetic theory has been proposed by Hermisson and Wagner (2004), who suggest that the increase in genetic variance V_G after the change in environmental conditions or genetic background is a generic property of the population, with no need to introduce canalization (Waddington 1957). The theory appears simple. Under mutation–selection balance (MSB), the mutant alleles are at a selective disadvantage and there is a negative correlation between frequencies and effects of mutations: mutant alleles of small effects on the trait segregate at intermediate frequencies. After the change in genetic or environmental background, gene effects consequently change due to G × E interaction or epistasis, which reduces the negative correlation because genes that were previously of small effects and at intermediate frequencies may now have large effects. That is, the frequencies of alleles are determined by the previous MSB, while their new effects are at least partly determined by the new conditions. The genetic variance will therefore increase.Hermisson and Wagner (2004) found that the predicted increase in genetic variance can be substantial; however, the predicted increase is highly sensitive to the population size and can increase without bound with increasing population size (see their Figure 2 and Equation 16). Genetic variance would enlarge with the population size within a small population (Lynch and Hill 1986; Weber and Diggins 1990), but becomes insensitive to the population size within large populations (Falconer and Mackay 1996, Chap. 20). Hence the unbounded increase under the novel environmental condition appears to us as a downside of their theory, even though the predicted increase can be reduced if the changed environmental condition is not novel but there is previous adaptation to it (see their Figure 3).Open in a separate windowFigure 2.—Influence of the pleiotropic effect (s_p) on the increase of genetic variance Δ_G in units of the interaction parameter ξ for a “typical” situation with strength of stabilizing selection ω² = 0.1μ², mutation rate λ = 0.1 per haploid genome per generation, and population size N_e = 10⁶. The allelic pleiotropic effect on fitness and its variance effect on the trait independently follow gamma distributions with shape parameters β_s and β_v, respectively. The mean of a² across loci is E(v) = E(a²) = 10⁻⁴μ².Open in a separate windowOpen in a separate windowFigure 3.—Influence of shapes of distributions of mutational effects on (a) the variances at mutation–selection balance and (b) their increases after the genetic or environmental change. The squares represent the genetic variance and its increase and the triangles the environmental variance and its increase. The mutation rate is λ= 0.1 per haploid genome per generation, the population size is N_e = 10⁹, and the strength of real stabilizing selection is ω² = 0.1μ². Allelic effects on trait value (a), fitness (s), and residual variance (b) are assumed to be independently distributed such that v = a² follows a gamma () distribution with mean 10⁻⁴μ², s follows gamma (β_s) with mean s_p = 0.05, and b follows gamma (β_b) with mean 10⁻⁴μ².The basic model that Hermisson and Wagner (2004) employed is that the quantitative trait is under real stabilizing selection and mutant alleles have effects on the focal trait only by changing its so-called locus genetic variance. At the mutation–real stabilizing selection balance, some mutants can segregate at intermediate frequencies because of their small effects and therefore weak selection; and there are more such mutants the more strongly leptokurtic is the distribution of effects at individual loci. The unbounded increase of Hermisson and Wagner (2004) results from such a gene-frequency distribution; but it has been shown (see Barton and Turelli 1989; Falconer and Mackay 1996; Lynch and Walsh 1998) that solely stabilizing selection, whether modeled with a Gaussian (Kimura 1965) or a house of-cards approximation (Turelli 1984) or even the generalized form of Hermisson and Wagner (2004) (i.e., their Equation 14), cannot provide a satisfactory explanation for the high levels of genetic variance observed in natural populations under realistic values of mutation and selection parameters.A common observation is that one trait is controlled by many genes and one gene can influence many traits; i.e., pleiotropy is ubiquitous (Barton and Turelli 1989; Barton and Keightley 2002; Mackay 2004; Ostrowski et al. 2005). Recent detailed studies suggest that pleiotropy calculated as the number of phenotypic traits affected varies considerably among quantitative trait loci (QTL) (Cooper et al. 2007; Albert et al. 2008; Kenney-Hunt et al. 2008; Wagner et al. 2008). Such pleiotropic effects must influence the magnitude of the variance. Though some genes have little effect on the focal trait, they almost certainly affect other traits and therefore are not neutral. The inclusion of pleiotropic effects on fitness strengthens the overall selection on mutant alleles and, assuming such pleiotropic effects are mainly deleterious, maintains them at low frequencies. The genetic variance for a trait is therefore likely to be maintained at lower levels than that under only real stabilizing selection on the trait alone (Tanaka 1996). Although the gene-frequency distribution is much more extreme under this joint model, the relevant rate of mutation is genomewide and hence is much larger than that where mutation affects only the focal trait as is assumed in the real stabilizing selection model (Turelli 1984; Falconer and Mackay 1996). Taking into account empirical knowledge of mutation parameters, a combination of both pleiotropic and real stabilizing selection appears to be a plausible mechanism for the maintenance of quantitative genetic variance (Zhang et al. 2004). If pleiotropic selection is much stronger than real stabilizing selection, the association between frequency and effect of mutant alleles is weaker than that for a real stabilizing selection model. Further, if overall selection is stronger than recurrent mutation, the frequency distribution of mutant alleles will be extreme. Under those situations, the increase of genetic variance after the genetic or environmental change will be kept at lower levels than that of Hermisson and Wagner (2004), and hence the unbounded increase could be avoided.Further, Hermisson and Wagner (2004) assume that the environmental variance is not under genetic control (i.e., the variance of phenotypic value given genotypic value is the same for all genotypes) and therefore is not subject to change. This assumption conflicts with the increasingly accumulating empirical data that indicate otherwise (Zhang and Hill 2005; Mulder et al. 2007 for reviews). Direct experimental evidence is available that mutation can directly affect environmental variance, V_E (Whitlock and Fowler 1999; Mackay and Lyman 2005), and Baer (2008) provides what is perhaps the first clear demonstration that mutations increase environmental variances, on the basis of data for body size and productivity of Caenorhabditis elegans, and finds that the magnitudes of the increases are of the same order as those in the genetic variance.As real stabilizing selection on phenotype favors genotypes possessing low V_E (Gavrilets and Hastings 1994; Zhang and Hill 2005), a mutant that contributes little to V_E is more favored by stabilizing selection than one that contributes a lot. With all else being the same, mutants with small effect on V_E thus segregate at relatively high frequencies at MSB. That is, there is a negative correlation between the effect on V_E and the frequency of mutant genes. After the genetic or environmental change, some mutants that were previously of small effects on V_E have large effects due to G × E interaction or epistasis while their frequencies remain roughly the same as in the previous MSB. This certainly increases environmental variance.In this note, we first assume that mutant alleles can affect only the mean value of a focal quantitative trait and otherwise affect fitness through their pleiotropic effects (Zhang et al. 2004) and try to answer the following questions: How will the conclusion of Hermisson and Wagner (2004) be affected by taking into account the pleiotropic effect of mutants? Can the “unbounded increase” be avoided? We then further assume that mutant alleles can also directly affect the environmental variance of the focal trait (Zhang and Hill 2008) and investigate how both V_G and V_E change following the genetic or environmental change in the population.     

A New Family of Ty1-copia-Like Retrotransposons Originated in the Tomato Genome by a Recent Horizontal Transfer Event

The use of β-lactam antibiotics has led to the evolution and global spread of a variety of resistance mechanisms, including β-lactamases, a group of enzymes that degrade the β-lactam ring. The evolution of increased β-lactam resistance was studied by exposing independent lineages of Salmonella typhimurium to progressive increases in cephalosporin concentration. Each lineage carried a β-lactamase gene (bla_TEM-1) that provided very low resistance. In most lineages, the initial response to selection was an amplification of the bla_TEM-1 gene copy number. Amplification was followed in some lineages by mutations (envZ, cpxA, or nmpC) that reduced expression of the uptake functions, the OmpC, OmpD, and OmpF porins. The initial resistance provided by bla_TEM-1 amplification allowed the population to expand sufficiently to realize rare secondary point mutations. Mathematical modeling showed that amplification often is likely to be the initial response because events that duplicate or further amplify a gene are much more frequent than point mutations. These models show the importance of the population size to appearance of later point mutations. Transient gene amplification is likely to be a common initial mechanism and an intermediate in stable adaptive improvement. If later point mutations (allowed by amplification) provide sufficient adaptive improvement, the amplification may be lost.THE extensive use of β-lactam antibiotics has led to the evolution and spread of many chromosomal-, plasmid-, and transposon-borne resistance mechanisms (Livermore 1995; Weldhagen 2004). Prominent among these mechanisms is a class of enzymes, β-lactamases, that hydrolyze the β-lactam ring (Ambler 1980; Poole 2004). TEM-1 β-lactamase, encoded by the bla_TEM-1 gene, hydrolyzes both penicillins and early cephalosporins (Matagne et al. 1990). As bacteria developed resistance, stable extended-spectrum cephalosporins (ESCs) were introduced, leading to evolution of TEM sequence variants with improved ESC hydrolysis (Petrosino et al. 1998). Resistance to β-lactams can also result from mutations that reduce levels of outer membrane proteins involved in uptake, altered target proteins (penicillin-binding proteins) to reduce β-lactam binding, or increased expression of efflux pumps that export the antibiotics (Poole 2004; Martínez-Martínez 2008; Zapun et al. 2008).Resistance to β-lactam antibiotics is linearly correlated with the lactamase level over a large range (Nordström et al. 1972) and resistance to β-lactam antibiotics can be provided by increasing enzyme levels. An early illustration of this process is the finding that Escherichia coli can develop ampicillin resistance by amplifying its ampC gene (Edlund and Normark 1981). Similar amplification has been observed in both eubacteria and eukaryotes (Craven and Neidle 2007; Wong et al. 2007) in response to various selective pressures, including antibiotics (Andersson and Hughes 2009; Sandegren and Andersson 2009). In an unselected bacterial population, the frequency of cells with a duplication of any specific chromosomal region ranges between 10⁻² and 10⁻⁵ depending on the region (Anderson and Roth 1981), whereas a point mutation in that gene is expected to be carried by perhaps 1 cell in 10⁷–10⁸ (Hudson et al. 2002). Thus, the rate of duplication formation is ∼10⁻⁵/cell/division and further increases ∼0.01/cell/division (Pettersson et al. 2008) while the base substitution rate is ∼10⁻¹⁰/cell/division/base pair (Hudson et al. 2002). Thus, it is apparent that variants with an increased level of any enzyme activity are more likely to owe the increase to a gene copy number change than to a point mutation. Furthermore, because of the high intrinsic instability of tandem amplifications, haploid segregants are expected to take over the population when the selection pressure is released (Pettersson et al. 2008).To examine the importance of gene amplification in bacterial adaptation to cephalosporins, several independent Salmonella typhimurium lineages carrying the bla_TEM-1 gene were allowed to develop resistance to progressively increased concentrations of cephalothin (a first-generation cephalosporin) and cefaclor (a second-generation cephalosporin). As these lineages developed resistance to higher antibiotic levels, amplification of the bla_TEM-1 gene was the primary and most common resistance mechanism, which in some cases was followed by acquisition of rare point mutations that provided stable resistance.     

Barley beta‐glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H₂O₂-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O₂⁻) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O₂⁻, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure.     

Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon

1. Human uterine cervical stroma was found to contain a Ca²⁺-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×10⁵ by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.     

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

Isolation of active mitochondria from tomato fruit

An improved method for isolating mitochondria from tomato fruit (Lycopersicon esculentum Mill.) is described. The fruit is chilled, and the tissue of the fruit wall cut by hand into very thin slices with a razor blade while immersed in a buffer containing 0.4 m sucrose, 2 mm MgCl₂, 8 mm EDTA, 4 mm cysteine, 10 mm KCl, 0.5 mg per ml bovine serum albumin 50 mm tris-HCl, pH 7.6. The pH is monitored and kept within the range of 7.0 to 7.2 by dropwise addition of 1 n KOH during cutting. The tissue is strained through 8 layers of cheesecloth and centrifuged at 2000 × g for 15 minutes. The supernatant is then centrifuged at 11,000 × g for 20 minutes, and the sediment is washed once with a medium containing 0.4 m sucrose, 10 mm KCl, 1 mm MgCl₂, 10 mm tris-HCl, 10 mm KH₂PO₄ and bovine serum albumin (0.5 mg per ml), pH 7.2. Electron microscope studies show that this method gives homogeneous, relatively intact mitochondria; they have a higher respiratory control ratio than those reported by other workers. The method was also tested successfully on fruits of cantaloupe and `Honey Dew' melon.     

Crystal structure of a zinc-dependent D-serine dehydratase from chicken kidney

d-Serine is a physiological co-agonist of the N-methyl-d-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, d-amino acid oxidase degrades d-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a d-serine dehydratase degrades d-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated d-threonine aldolases, which are fold-type III pyridoxal 5′-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of d-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography. The enzyme activity on d-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn²⁺. None of the reaction products that would be expected from side reactions of the PLP-d-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity. We have determined the first crystal structure of the d-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His³⁴⁷ and Cys³⁴⁹ is located near the PLP-Lys⁴⁵ Schiff base. A theoretical model of the enzyme-d-serine complex suggested that the hydroxyl group of d-serine directly coordinates the zinc ion, and that the ϵ-NH₂ group of Lys⁴⁵ is a short distance from the substrate Cα atom. The α-proton abstraction from d-serine by Lys⁴⁵ and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.     

Potassium fluxes in dialyzed squid axons

Measurements have been made of K influx in squid giant axons under internal solute control by dialysis. With [ATP]_i = 1 µM, [Na]_i = 0, K influx was 6 ± 0.6 pmole/cm² sec; an increase to [ATP]_i = 4 mM gave an influx of 8 ± 0.5 pmole/cm² sec, while [ATP]_i 4, [Na]_i 80 gave a K influx of 19 ± 0.7 pmole/cm² sec (all measurements at ∼16°C). Strophanthidin (10 µM) in seawater quantitatively abolished the ATP-dependent increase in K influx. The concentration dependence of ATP-dependent K influx on [ATP]_i, [Na]_i, and [K]_o was measured; an [ATP]_i of 30 µM gave a K influx about half that at physiological concentrations (2–3 mM). About 7 mM [Na]_i yielded half the K influx found at 80 mM [Na]_i. The ATP-dependent K influx responded linearly to [K]_o from 1–20 mM and was independent of whether Na, Li, or choline was the principal cation of seawater. Substances tested as possible energy sources for the K pump were acetyl phosphate, phosphoarginine, PEP, and d-ATP. None was effective except d-ATP and this substance gave 70% of the maximal flux only when phosphoarginine or PEP was also present.     

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in l-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by l-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with l-arginine bound and in the active R-state complexed with CoA and l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of l-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by ∼10 Å and decreases its height by ∼20Å_. AAK dimers move 5Å outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by ∼4°. The NAT domains rotate ∼109° relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the l-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.l-Arginine biosynthesis in most micro-organisms and plants involves the initial acetylation of l-glutamate by N-acetylglutamate synthase (NAGS, EC 2.3.1.1)² to produce N-acetylglutamate (NAG). NAG is then converted by NAG kinase (NAGK, EC 2.7.2.8) to NAG-phosphate and subsequently to N-acetylornithine (1, 2). Two alternative reactions are used to remove the acetyl group from acetylornithine. The linear pathway uses N-acetylornithine deacetylase (EC 3.5.1.16) to catalyze the metal-dependent hydrolysis of the acetyl group to form l-ornithine and acetate, whereas the acetyl recycling pathway transfers the acetyl group from N-acetylornithine to l-glutamate, producing l-ornithine and NAG. This reaction is catalyzed by ornithine acetyltransferase (EC 2.3.1.35).In the linear pathway, NAGS is the only target of feedback inhibition by l-arginine. In contrast, in the acetyl cycling pathway l-arginine may inhibit NAGS and NAGK or ornithine acetyltransferase (3). Structure determinations of l-arginine-insensitive (4) and l-arginine-sensitive NAGKs (5) provided insights into the structural basis of l-arginine inhibition of NAGK. l-Arginine-insensitive Escherichia coli (ec) NAGK is a homodimer (4), whereas l-arginine-sensitive NAGKs from Thermotoga maritima (tm) and Pseudomonas aeruginosa (pa) are hexamers formed by pair-wise interlacing of the N-terminal helices of three ecNAGK-like dimers, to create a second type of dimer interface. l-Arginine binding to a site close to the C terminus induces global conformational changes that expands the ring by ∼8 Å and decreases the tilt of the ecNAGK-like dimers relative to the plane of the ring by ∼6°. The inhibition mechanism was proposed to involve the enlargement of an active site located close to the l-arginine-binding site.Because of the sequence similarity between NAGK and NAGS, it was speculated that they may have similar l-arginine-binding sites and hexameric ring structures (5). However, our recent structural determination of NAGS from Neisseria gonorrhoeae (ng) revealed the active site to be located in the NAT domain, >25 Å away from the proposed l-arginine-binding site (6). Therefore, the allosteric mechanism of NAGS is likely to be different from that of l-arginine-sensitive NAGKs. Here we compare the structures of ngNAGS in the inactive T-state with l-arginine bound and in the R-state complexed with CoA and l-glutamate and determine the structural basis for the allosteric inhibition of NAGS by l-arginine.     

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K_m 0.042mm), 2.0mm-ATP (K_m 0.19mm) and 8mm-Mg²⁺ at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (K_m 0.004mm), 1.5mm-ATP (K_m 0.12mm) and 2mm-Mg²⁺. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.     

Decreased K+ conductance produced by Ba++ in frog sartorius fibers

The action of Ba⁺⁺ on membrane potential (E_m) and resistance (R_m) of frog (R. pipiens) sartorius fibers was studied. In normal Cl^- Ringer''s, Ba⁺⁺ (<9 mM) did not depolarize or induce contractions, but increased R_m slightly above the control value of 3.8 ± 0.6 KΩ-cm². In Cl^--free Ringer''s (methane sulfonate) R_m was 28.8 ± 2.8 KΩ-cm², and low concentrations of Ba⁺⁺ (0.05–5.0 mM) depolarized and induced spontaneous contractions (fibrillation), even in tetrodotoxin. To stop disturbance of the microelectrodes, contractions were prevented by using two Cl^--free solutions: (a) twice hypertonic with sucrose (230 mM), or (b) high K⁺ (83 mM) partially replacing Na⁺. In the hypertonic solution, the fiber diameters decreased, E_m increased slightly, and R_m decreased to 9.0 ± 0.6 KΩ-cm² (perhaps due to swelling of sarcotubules). Ba⁺⁺ (0.5 mM) rapidly increased R_m to 31.3 ± 3.8, decreased E_m (e.g., to -30 mv), and induced spontaneous "action potentials;" Sr⁺⁺ had no effect. In the high K⁺ solution, the fibers were nearly completely depolarized, and R_m was decreased markedly to 1.5 ± 0.2 KΩ-cm²; Ba⁺⁺ increased R_m to 6.7 ± 0.5 KΩ-cm². The Ba⁺⁺ actions usually began within 0.5 min and reached a maximum within 5 min. Addition of SO₄ ⁼, to precipitate the Ba⁺⁺, rapidly reversed the increase in R_m. Ba⁺⁺ must act by decreasing K⁺ conductance (g_K). In Cl^- Ringer''s, the high g_Cl/g_K ratio masked the effect of Ba⁺⁺ on g_K. Thus, small concentrations of Ba⁺⁺ specifically and rapidly decrease g_K.     

A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase

Pyranose 2-oxidase (P2O) catalyzes the oxidation by O² of d-glucose and several aldopyranoses to yield the 2-ketoaldoses and H²O². Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr¹⁶⁹ forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O·sugar complex. Transient kinetics of wild-type (WT) and Thr¹⁶⁹ → S/N/G/A replacement variants show that d-Glc binds to T169S, T169N, and WT with the same K_d (45–47 mm), and the hydride transfer rate constants (k^red) are similar (15.3–9.7 s⁻¹ at 4 °C). k^red of T169G with d-glucose (0.7 s⁻¹, 4 °C) is significantly less than that of WT but not as severely affected as in T169A (k^red of 0.03 s⁻¹ at 25 °C). Transient kinetics of WT and mutants using d-galactose show that P2O binds d-galactose with a one-step binding process, different from binding of d-glucose. In T169S, T169N, and T169G, the overall turnover with d-Gal is faster than that of WT due to an increase of k^red. In the crystal structure of T169S, Ser¹⁶⁹ Oγ assumes a position identical to that of Oγ1 in Thr¹⁶⁹; in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr¹⁶⁹ side chain are required for intermediate stabilization.     

Ibuprofen Impairs Allosterically Peroxynitrite Isomerization by Ferric Human Serum Heme-Albumin

Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO₃⁻ catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 °C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO₂; the values of the second order catalytic rate constant (k_on) are 4.1 × 10⁵ and 4.5 × 10⁵ m⁻¹ s⁻¹, respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of k_on (pK_a = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO₂. The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO₂, ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 × 10⁻⁴ and 9.7 × 10⁻⁴ m. Under conditions where [ibuprofen] is ≫L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role of HSA-heme-Fe(III) in vivo.     

Rational design and characterization of D-Phe-Pro-D-Arg-derived direct thrombin inhibitors

The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both l- and d-amino acids, with the general sequence d-Phe(P3)-Pro(P2)-d-Arg(P1)-P1′-CONH₂. The P1′ position was scanned with l- and d-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1′ position. The lead tetrapeptide, d-Phe-Pro-d-Arg-d-Thr-CONH₂, competitively inhibits α-thrombin''s cleavage of the S2238 chromogenic substrate with a K_i of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1′ l-isoleucine (fPrI), l-cysteine (fPrC) or d-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the d-Arg residue in position P1 and thrombin are similar to those observed for the l-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 d-Arg and a bulkier P1′ residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the d-stereochemistry of the residues at positions P1 and P1′.     

Chronic Exposure to Excess Nutrients Left-shifts the Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic β-Cells

Hyperinsulinemia (HI) is elevated plasma insulin at basal glucose. Impaired glucose tolerance is associated with HI, although the exact cause and effect relationship remains poorly defined. We tested the hypothesis that HI can result from an intrinsic response of the β-cell to chronic exposure to excess nutrients, involving a shift in the concentration dependence of glucose-stimulated insulin secretion. INS-1 (832/13) cells were cultured in either a physiological (4 mm) or high (11 mm) glucose concentration with or without concomitant exposure to oleate. Isolated rat islets were also cultured with or without oleate. A clear hypersensitivity to submaximal glucose concentrations was evident in INS-1 cells cultured in excess nutrients such that the 25% of maximal (S_0.25) glucose-stimulated insulin secretion was significantly reduced in cells cultured in 11 mm glucose (S_0.25 = 3.5 mm) and 4 mm glucose with oleate (S_0.25 = 4.5 mm) compared with 4 mm glucose alone (S_0.25 = 5.7 mm). The magnitude of the left shift was linearly correlated with intracellular lipid stores in INS-1 cells (r² = 0.97). We observed no significant differences in the dose responses for glucose stimulation of respiration, NAD(P)H autofluorescence, or Ca²⁺ responses between left- and right-shifted β-cells. However, a left shift in the sensitivity of exocytosis to Ca²⁺ was documented in permeabilized INS-1 cells cultured in 11 versus 4 mm glucose (S_0.25 = 1.1 and 1.7 μm, respectively). Our results suggest that the sensitivity of exocytosis to triggering is modulated by a lipid component, the levels of which are influenced by the culture nutrient environment.