Molecular Cloning,Structural Modeling and the Production of Soluble Triple-Mutated Diphtheria Toxoid (K51E/G52E/E148K) Co-expressed with Molecular Chaperones in Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However, it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97.33 ± 17.47 μg/ml) under the optimal condition (induction with 0.1 mM IPTG at 20 °C for 24 h). Cells containing pG-Tf2, expressing trigger factor and GroEL-GroES, accumulated the highest amount of soluble CRM197EKTrxHis at 111.24 ± 10.40 μg/ml after induction for 24 h at 20 °C. The soluble CRM197EKTrxHis still possesses nuclease activity and completely digest λDNA at 25 and 37 °C with 8- and 4-h incubation, respectively. Molecular modeling of diphtheria toxin, CRM197 and CRM197EK indicated that substitutions of two amino acids (K51E/E148K) may cause poor NAD binding, consistent with the lack of toxicity. Therefore, CRM197EK might be used as a new potential carrier protein. However, further in vivo study is required to confirm its roles as functional carrier protein in conjugate vaccines.     

Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of <Emphasis Type="Italic">Hypericum hirsutum</Emphasis> and <Emphasis Type="Italic">Hypericum maculatum</Emphasis>

We investigated the effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 6-γ,γ-dimethylallylaminopurine (2iP), thidiazuron (TDZ) and α-naphthaleneacetic acid (NAA)], modified Murashige and Skoog (MS) medium containing 10 mM NH₄ ⁺ and 5 mM NO₃ ⁻ and supplemented with 2iP, BA, Kin and NAA (MSM medium), and two elicitors [jasmonic acid (JA), and salicylic acid (SA)], on plant growth and accumulation of hypericins (hypericin and pseudohypericin) and hyperforin in shoot cultures of Hypericum hirsutum and H. maculatum. Our data suggested that culture of shoots on MS medium supplemented with BA (0.4 mg l⁻¹) or Kin (0.4 mg l⁻¹) enhanced production of hypericins in H. maculatum and hyperforin in H. hirsutum. Hypericins and hyperforin concentrations decreased in both species when TDZ (0.4 mg l⁻¹) was added to the MS medium. Also, TDZ induced hyperhydric malformations and necrosis of regenerated shoots. Cultivation of H. maculatum on MSM medium resulted in approximately twofold increased production of hypericins compared to controls, and the growth of H. hirsutum shoots on the same medium led to a 6.16-fold increase in hyperforin production. Of the two elicitors, SA was more effective in stimulating the accumulation of hypericins. At 50 μM, SA enhanced the production of hypericin (7.98-fold) and pseudohypericin (13.58-fold) in H. hirsutum, and, at 200 μM, enhanced the production of hypericin (2.2-fold) and pseudohypericin (3.94-fold) in H. maculatum.     

Acid dissociation constants of diphytanylglycerolphosphorylglycerol-methylphosphate, and diphytanylglycerolphosphorylglycerophosphate and its deoxy analog

Acid dissociation constants of 2,3-diphytanyl-sn-glycero-1-phosphoryl-sn-3′-glycero-1′-methylphosphate (PGP-Me), the major phospholipid in extreme halophiles (Halobacteriaceae), and of the demethylated 2,3-diphytanyl-sn-glycero-1-phosphoryl-sn-3′-glycero-1′-phosphate (PGP) and its deoxy analog 2,3-diphytanyl-sn-glycero-1-phosphoryl-1′-(1′,3′-propanediol-3′-phosphate) (dPGP), were calculated by an original mathematical procedure from potentiometric titration data in methanol/water (1:1, v/v) and found to be as follows: for PGP-Me (and presumably PGP), pK₁=3.00 and pK₂=3.61; for PGP, pK₃=11.12; and for dPGP, pK₁=2.72, pK₂=4.09, and pK₃=8.43. High-resolution ³¹P NMR spectra of intact PGP-Me in benzene/methanol or in aqueous dispersion showed two resonances corresponding to the two P-OH groups, each of which exhibited a chemical shift change in the pH range 2.0–4.5, corresponding to acid dissociation constants pK₁=pK₂=3.2; no further ionization (pK₃) was detected at higher pH values in the range 5–12. The present results show that PGP-Me titrates as a dibasic acid in the pH range 2–8, but above pH 8, it titrates as a tribasic acid, presumably PGP, formed by hydrolysis of the methyl group during the titration with KOH. Calculation of the concentrations of the ionic molecular species of PGP-Me, PGP and dPGP as a function of pH showed that the dianionic species predominate in the pH range 5–9, covering the optimal pH for growth of Halobacteriaceae. The results are consistent with the concept that the hydroxyl group of the central glycerol moiety in PGP-Me and PGP is involved in the formation of an intramolecular hydrogen-bonded cyclic structure of the polar headgroup, which imparts greater stability to the dianionic form of PGP-Me and PGP in the pH range 5–9 and facilitates lateral proton conduction by a process of diffusion along the membrane surface of halobacterial cells.     

A multistudy analysis reveals that evoked pain intensity representation is distributed across brain systems

Information is coded in the brain at multiple anatomical scales: locally, distributed across regions and networks, and globally. For pain, the scale of representation has not been formally tested, and quantitative comparisons of pain representations across regions and networks are lacking. In this multistudy analysis of 376 participants across 11 studies, we compared multivariate predictive models to investigate the spatial scale and location of evoked heat pain intensity representation. We compared models based on (a) a single most pain-predictive region or resting-state network; (b) pain-associated cortical–subcortical systems developed from prior literature (“multisystem models”); and (c) a model spanning the full brain. We estimated model accuracy using leave-one-study-out cross-validation (CV; 7 studies) and subsequently validated in 4 independent holdout studies. All spatial scales conveyed information about pain intensity, but distributed, multisystem models predicted pain 20% more accurately than any individual region or network and were more generalizable to multimodal pain (thermal, visceral, and mechanical) and specific to pain. Full brain models showed no predictive advantage over multisystem models. These findings show that multiple cortical and subcortical systems are needed to decode pain intensity, especially heat pain, and that representation of pain experience may not be circumscribed by any elementary region or canonical network. Finally, the learner generalization methods we employ provide a blueprint for evaluating the spatial scale of information in other domains.

Is pain represented by a single brain area or network, spanning multiple systems or distributed throughout the brain? fMRI brain decoding in a large multi-study dataset shows that multiple cortical and subcortical systems are needed to decode pain intensity; the approach is novel and can characterize scale of representation across diverse brain processes.     

Diffusional water permeability of mammalian red blood cells

An extensive programme of comparative nuclear magnetic resonance measurements of the membrane diffusional permeability for water (P_d) and of the activation energy (E_a,d) of this process in red blood cells (RBCs) from 21 mammalian species was carried out. On the basis of P_d, these species could be divided into three groups. First, the RBC's from humans, cow, sheep and “large” kangaroos (Macropus giganteus and Macropus rufus) had P_d values 5 × 10⁻³ cm/s at 25°C and 7 × 10⁻³ cm/s at 37°C. The RBCs from other marsupial species, mouse, rat, guinea pig and rabbit, had P_d values roughly twice higher, whereas echidna RBCs were twice lower than human RBCs. The value of E_a,d was in most cases correlated with the values of P_d. A value of E_a,d -26 kJ/mol was found for the RBCs from humans and the species having similar P_d values. Low values of E_a,d (ranging from 15 to 22 kJ/mol) appeared to be associated with relatively high values of P_d. The highest value of E_a,d (33 kJ/mol) was found in echidna RBCs. This points to specialized channels for water diffusion incorporated in membrane proteins; a relatively high water permeability of the RBC membrane could be due to a greater number of channel proteins. There are, however, situations where a very high water permeability of RBCs is associated with a high value of E_a,d (above 25 kJ/mol) as in the case of RBCs from mouse, rat and tree kangaroo. Moreover, it was found that P_d in different species was positively correlated to the RBC membrane phosphatidylcholine and negatively correlated to the sphingomyelin content. This suggests that in addition to the number of channel proteins, other factors are involved in the water permeability of the RBC membrane.     

"Wide-open" 1.3 A structure of a multidrug-resistant HIV-1 protease as a drug target

This report examines structural changes in a highly mutated, clinical multidrug-resistant HIV-1 protease, and the crystal structure has been solved to 1.3 A resolution in the absence of any inhibitor. This protease variant contains codon mutations at positions 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90 that confer resistance to protease inhibitors. Major differences between the wild-type and the variant include a structural change initiated by the M36V mutation and amplified by additional mutations in the flaps of the protease, resulting in a "wide-open" structure that represents an opening that is 8 A wider than the "open" structure of the wild-type protease. A second structural change is triggered by the L90M mutation that results in reshaping the 23-32 segment. A third key structural change of the protease is due to the mutations from longer to shorter amino acid side chains at positions 82 and 84.