Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

Ex vivo protective effects of nicotinamide and 3‐aminobenzamide on rat synaptosomes treated with Aβ(1–42)

Alzheimer's disease (AD) is the most common form of dementia and is characterized by the presence of senile plaques and neurofibrillary tangles, along with synaptic loss. The underlying mechanisms of AD are not clarified yet, but oxidative stress and mitochondrial dysfunction are important factors. Overactivation of poly(adenosine diphosphate ribose) polymerase‐1 (PARP‐1) enzyme has been known to cause neuroinflammation and cell death in neurodegenerative processes. The aim of the present study was to investigate the protective effects of the PARP‐1 inhibitors, 3‐aminobenzamide (3‐AB) and nicotinamide (NA), against amyloid β peptide (1–42) (Aβ(1–42))‐induced oxidative damage and mitochondrial reduction capacity on isolated synaptosomes. Rats were injected intraperitoneally with 3‐AB (30–100 mg kg^?1), NA (100–500 mg kg^?1) or with saline for 7 days. Synaptosomes were incubated with 10–30 μM Aβ(1–42) or saline for 6 h at 37 °C. Ex vivo Aβ(1–42) treatment significantly induced oxidative stress and mitochondrial dysfunction in synaptosomes of the saline group, while synaptosomes of 3‐AB and NA groups showed significant decreases in lipid peroxidation, reactive oxygen species production and protein oxidation. Moreover, both NA and 3‐AB were able to improve the mitochondrial reduction capacity against Aβ(1–42). These data suggest that NA and 3‐AB may have protective effects in neurodegenerative processes because of the reduced levels of oxidative stress and the improvement of mitochondrial function. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis of a New Series of 1H‐Imidazol‐1‐yl Substituted 8‐Phenylxanthines as Adenosine Receptor Ligands

A new series of 1H‐imidazol‐1‐yl substituted 8‐phenylxanthine analogs has been synthesized to study the effects of the imidazole group on the binding affinity of compounds for adenosine receptors. Competition binding studies of these compounds were carried out in vitro with human cloned receptors using [³H]DPCPX and [³H]ZM 241385 as radioligands at A₁ and A_2A adenosine receptors, respectively. The effect of the substitution pattern of the (imidazolyl)alkoxy group on various positions of the phenyl ring at C(8) was also studied. The xanthine derivatives displayed varying degrees of affinity and selectivity towards A₁ and A_2A receptor subtypes despite a common but variedly substituted Ar C(8).     

Hopeahainol A attenuates memory deficits by targeting β‐amyloid in APP/PS1 transgenic mice

Increasing evidence demonstrates that amyloid beta (Aβ) elicits mitochondrial dysfunction and oxidative stress, which contributes to the pathogenesis of Alzheimer's disease (AD). Identification of the molecules targeting Aβ is thus of particular significance in the treatment of AD. Hopeahainol A (HopA), a polyphenol with a novel skeleton obtained from Hopea hainanensis, is potentially acetylcholinesterase‐inhibitory and anti‐oxidative in H₂O₂‐treated PC12 cells. In this study, we reported that HopA might bind to Aβ_1–42 directly and inhibit the Aβ_1–42 aggregation using a combination of molecular dynamics simulation, binding assay, transmission electron microscopic analysis and staining technique. We also demonstrated that HopA decreased the interaction between Aβ_1–42 and Aβ‐binding alcohol dehydrogenase, which in turn reduced mitochondrial dysfunction and oxidative stress in vivo and in vitro. In addition, HopA was able to rescue the long‐term potentiation induction by protecting synaptic function and attenuate memory deficits in APP/PS1 mice. Our data suggest that HopA might be a promising drug for therapeutic intervention in AD.     

Exploration of multi‐target effects of 3‐benzoyl‐5‐hydroxychromen‐2‐one in Alzheimer’s disease cell and mouse models

Microtubule‐associated protein Tau, abundant in the central nervous system (CNS), plays crucial roles in microtubule assembly and stabilization. Abnormal Tau phosphorylation and aggregation are a common pathogenic hallmark in Alzheimer's disease (AD). Hyperphosphorylation of Tau could change its conformation and result in self‐aggregation, increased oxidative stress, and neuronal death. In this study, we examined the potential of licochalcone A (a natural chalcone) and five synthetic derivatives (LM compounds) for inhibiting Tau misfolding, scavenging reactive oxygen species (ROS) and providing neuroprotection in human cells expressing proaggregant ΔK280 Tau_RD‐DsRed. All test compounds were soluble up to 100 μM in cell culture media and predicted to be orally bioavailable and CNS‐active. Among them, licochalcone A and LM‐031 markedly reduced Tau misfolding and associated ROS, promoted neurite outgrowth, and inhibited caspase 3 activity in ΔK280 Tau_RD‐DsRed 293 and SH‐SY5Y cells. Mechanistic studies showed that LM‐031 upregulates HSPB1 chaperone, NRF2/NQO1/GCLC pathway, and CREB‐dependent BDNF/AKT/ERK/BCL2 pathway in ΔK280 Tau_RD‐DsRed SH‐SY5Y cells. Decreased neurite outgrowth upon induction of ΔK280 Tau_RD‐DsRed was rescued by LM‐031, which was counteracted by knockdown of NRF2 or CREB. LM‐031 further rescued the downregulated NRF2 and pCREB, reduced Aβ and Tau levels in hippocampus and cortex, and ameliorated cognitive deficits in streptozocin‐induced hyperglycemic 3 × Tg‐AD mice. Our findings strongly indicate the potential of LM‐031 for modifying AD progression by targeting HSPB1 to reduce Tau misfolding and activating NRF2 and CREB pathways to suppress apoptosis and promote neuron survival, thereby offering a new drug development avenue for AD treatment.     

Promoting α‐secretase cleavage of beta‐amyloid with engineered proteolytic antibody fragments

Deposition of beta‐amyloid (Aβ) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of Aβ levels by various therapeutic approaches is actively being pursued. A potentially non‐inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze Aβ at its α‐secretase site. We have previously identified a light chain fragment, mk18, with α‐secretase‐like catalytic activity, producing the 1–16 and 17–40 amino acid fragments of Aβ40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking α‐secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for Aβ. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the α‐secretase site of Aβ. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3‐ and 6‐fold increase in catalytic activity (k_cat/K_M) toward the synthetic Aβ substrate compared to the original scFv primarily due to an expected decrease in K_M rather than an increase in k_cat. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for Aβ. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble Aβ levels in vivo. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

Interaction of β‐cyclodextrin‐capped CdSe quantum dots with inorganic anions and cations

A facile method was developed for the preparation of water soluble β‐Cyclodextrin (β‐CD)‐modified CdSe quantum dots (QDs) (β‐CD‐QDs) by directly replacing the oleic acid ligands on the QDs surface with β‐CD in an alkaline aqueous solution. The as‐prepared QDs show good stability in aqueous solution for several months. Oxoanions, including phosphoric acid ion, sulphite acid ion and carbonic acid ion, affect the fluorescence of β‐CD‐QDs. Among them, H₂PO₄^– exhibited the largest quenching effect. For the polyprotic acids (HO)₃AO, the effect of acidic anions on the fluorescence of β‐CD‐QDs was in the order: monoanion (HO)₂AO₂^– > dianion (HO)AO₃^2– >> trianion AO₄^3–. After photoactivation for several days in the presence of anions at alkaline pH, the β‐CD‐QDs exhibited strong fluorescence emission. The effect of various heavy and transition metal ions on the fluorescence properties of the β‐CD‐QDs was investigated further. It was found that Ag⁺, Hg²⁺ and Co²⁺ have significant quenching effect on the fluorescence of the β‐CD‐QDs. The Stern–Volmer quenching constants increased in the order: Hg²⁺ < Co²⁺ <Ag⁺. The adsorption model of metal ions on β‐CD‐QDs was explored. Copyright © 2011 John Wiley & Sons, Ltd.     

The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63‐specific IFN‐γ‐secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA‐A*0201 restriction of ten predicted MPT63‐derived CD8 ⁺ T‐cell epitopes was assessed on the basis of T2 cell line and HLA‐A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN‐γ enzyme‐linked immunospot assay. It was found that five peptides bound to HLA‐A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA‐A*0201. Five immunogenic peptides (MPT63_18–26, MPT63_29–37, MPT63_20–28, MPT63_5–14 and MPT63_10–19) elicited large numbers of cytotoxic IFN‐γ‐secreting T cells in HLA‐A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA‐A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T‐SPOT.TB assay (based on ESAT‐6 and CFP‐10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T‐SPOT.TB assay reached 100%. These MPT63‐derived HLA‐A*0201‐restricted CD8 ⁺ T‐cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine.     

The N‐terminal tripeptide of insulin‐like growth factor‐I protects against β‐amyloid‐induced somatostatin depletion by calcium and glycogen synthase kinase 3β modulation

The protective effects of insulin‐like growth factor I on the somatostatin (SRIF) system in the temporal cortex after β‐amyloid (Aβ) injury may be mediated through its N‐terminal tripeptide glycine‐proline‐glutamate (GPE). GPE is cleaved to cyclo[Pro‐Gly] (cPG), a metabolite suggested to mediate in neuroprotective actions. We evaluated the effects of GPE and cPG in the temporal cortex of Aβ25–35‐treated rats on SRIF and SRIF receptor protein and mRNA levels, adenylyl cyclase activity, cell death, Aβ25–35 accumulation, cytosolic calcium levels ([Ca²⁺]_c) and the intracellular signaling mechanisms involved. GPE and cPG did not change Aβ25–35 levels, but GPE partially restored SRIF and SRIF receptor 2 protein content and mRNA levels and protected against cell death after Aβ25–35 insult, which was coincident with Akt activation and glycogen synthase kinase 3β inhibition. In addition, GPE displaced glutamate from NMDA receptors and blocked the glutamate induced rise in cytosolic calcium in isolated rat neurons and moderately increased Ca²⁺ influx per se. Our findings suggest that GPE, but not its metabolite, mimics insulin‐like growth factor I effects on the SRIF system through a mechanism independent of Aβ clearance that involves modulation of calcium and glycogen synthase kinase 3β signaling.     

Revealing interaction between sulfobutylether‐β‐cyclodextrin and reserpine by chemiluminescence and site‐directed molecular docking

The host–guest interaction between sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) and reserpine (RSP) is described using flow injection‐chemiluminescence (FI‐CL) and site‐directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE‐β‐CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ?I = 107.1lgC_RES + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5–106.1%. Using the proposed CL model, the binding constant (K_CD‐R) and the stoichiometric ratio of SBE‐β‐CD/RSP were calculated to be 7.4 × 10⁶ M^‐1 and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE‐β‐CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE‐β‐CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host–guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis,Characterization, and Anti‐Amoebic Activity of N‐(Pyrimidin‐2‐yl)benzenesulfonamide Derivatives

A new series of N‐(pyrimidin‐2‐yl)benzenesulfonamide derivatives, 3a – 3i and 4a – 4i , was synthesized from pyrimidin‐2‐amines, 2a – 2i , with the aim to explore their effects on in vitro growth of Entamoeba histolytica. The chemical structures of the compounds were elucidated by elemental analysis, FT‐IR, ¹H‐ and ¹³C‐NMR, and ESI mass‐spectral data. In vitro anti‐amoebic activity was evaluated against HM1 : IMSS strain of Entamoeba histolytica. The IC₅₀ values were calculated by using the double dilution method. The results were compared with the IC₅₀ value of the standard drug ‘metronidazole’. The selected compounds were tested for their cytotoxic activities by cell‐viability assay using H9C2 cardiac myoblasts cell line, and the results indicated that all the compounds displayed remarkable >80% viabilities to a concentration of 100 μg/ml.     

Synthesis of Novel Chiral Sulfonamide‐Bearing 1,2,4‐Triazole‐3‐thione Analogs Derived from D‐ and L‐Phenylalanine Esters as Potential Anti‐Influenza Agents

Novel enantiopure 1,2,4‐trizole‐3‐thiones containing a benzensulfonamide moiety were synthesized via multistep reaction sequence starting with D‐phenylalanine methyl ester and L‐phenylalanine ethyl ester as a source of chirality. The chemical structures of all compounds were characterized by elemental analysis, UV, IR, ¹H NMR, ¹³C NMR, 2D NMR (HETCOR), and mass spectral data. All compounds were tested in vitro antiviral activity against a broad variety of DNA and RNA viruses and in vitro cytostatic activity against murine leukemia (L1210), human T‐lymphocyte (CEM) and human cervix carcinoma (HeLa) cell lines. Although enantiopure 1,2,4‐triazole‐3‐thione analogs in (R) configuration emerged as promising anti‐influenza A H1N1 subtype in Madin Darby canine kidney cell cultures (MDCK), their enantiomers exhibited no activity. Especially compounds 18a , 21a , 22a , 23a , and 24a (EC₅₀: 6.5, 6.1, 2.4, 1.6, 1.7 μM, respectively) had excellent activity against influenza A H1N1 subtype compared to the reference drug ribavirin (EC₅₀: 8.0 μM). Several compounds have been found to inhibit proliferation of L1210, CEM and HeLa cell cultures with IC₅₀ in the 12–53 μM range. Compound 5a and 27a in (R) configuration were the most active compounds (IC₅₀: 12–22 μM for 5a and IC₅₀: 19–23 μM for 27a ). Chirality 28:495–513, 2016. © 2016 Wiley Periodicals, Inc.     

Chiral Discrimination of Ammonium Neurotransmitters by C3‐Symmetric Enantiopure Hemicryptophane Hosts

Enantiopure hemicryptophanes efficiently discriminate chiral ammonium neurotransmitters. The ephedrine and norephedrine molecules associate with hemicryptophane hosts to form 1:1 and 1:2 host‐guest complexes. Binding constants are determined by fitting the ¹H nuclear magnetic resonance (NMR) titration curves to give β₁ and β₂ values, which are used to characterize the diastereomeric and enantiomeric discriminating potentials of the hosts. Chirality 25:47–479, 2013. © 2013 Wiley Periodicals, Inc.     

Effective antimicrobial activity of Cbf‐K16 and Cbf‐A7A13 against NDM‐1‐carrying Escherichia coli by DNA binding after penetrating the cytoplasmic membrane in vitro

New Delhi metallo‐beta‐lactamase‐1(NDM‐1)‐carrying isolates, which are resistant to most clinical used antibiotics except for tigecycline and colistin, have been found worldwide. Cathelicidin‐BF (BF‐30) is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Cbf‐K₁₆ and Cbf‐A₇A₁₃ were obtained by mutating Lys¹⁶, Ala⁷, and Ala¹³ of BF‐30, respectively. To investigate their antimicrobial activities against NDM‐1 carrying bacteria, recombinant Escherichia coli BL21 (DE3)‐NDM‐1 with high NDM‐1 activity was constructed by inserting the Klebsiella pneumoniae NDM‐1 gene (GenBank accession no. HQ328085) into a pET28a vector and transforming it into E. coli BL21 (DE3). The peptides showed effective antimicrobial activities against NDM‐1‐carrying E. coli, and the minimum inhibitory concentrations of Cbf‐K₁₆ and Cbf‐A₇A₁₃ were only 4 and 8 µg/ml, whereas those of minimum bactericidal concentrations were 8 and 16 µg/ml, respectively. A time course experiment showed that colony forming unit counts rapidly decreased, and bacteria were thoroughly eliminated within 3 and 6 h by the Cbf‐K₁₆ and Cbf‐A₇A₁₃ treatments, respectively. The peptides penetrated the bacterial cell membrane and enabled β‐galactosidase leakage, and caused the cytoplasmic membrane to become permeable, and finally bound to the DNA. The genomic DNA of E. coli was completely unable to migrate on an agarose gel after Cbf‐K₁₆ treatment (8 µg/ml). These data demonstrated that Cbf‐K₁₆ and Cbf‐A₇A₁₃ possess effective antimicrobial activity against drug‐resistant strains, including NDM‐1 carrying E. coli BL21 (DE3)‐NDM‐1, by binding to DNA after penetrating the cytoplasmic membrane in vitro, which may have potential therapeutic value for the treatment of NDM‐1‐carrying bacterial infections. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis,binding affinities and metabolic stability of dimeric dermorphin analogs modified with β3‐homo‐amino acids

In this study, proteinogenic amino acids residues of dimeric dermorphin pentapeptides were replaced by the corresponding β³‐homo‐amino acids. The potency and selectivity of hybrid α/β dimeric dermorphin pentapeptides were evaluated by competetive receptor binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). Tha analog containing β³‐homo‐Tyr in place of Tyr (Tyr‐d ‐Ala‐Phe‐Gly‐β³‐homo‐Tyr‐NH‐)₂ showed good μ receptor affinity and selectivity (IC₅₀ = 0.302, IC₅₀ ratio μ/δ = 68) and enzymatic stability in human plasma. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Antibacterial Activity of Enrofloxacin and Ciprofloxacin Derivatives of β‐Octaarginine

β³‐Octaarginine chains were attached to the functional groups NH and CO₂H of the antibacterial fluoroquinolones ciprofloxacin (→ 1 ) and enrofloxacin (→ 2 ), respectively, in order to find out whether the activity increases by attachment of the polycationic, cell‐penetrating peptide (CPP) moiety. For comparison, simple amides, 3 – 5 , of the two antimicrobial compounds and β³‐octaarginine amide ( βR₈ ) were included in the antibacterial susceptibility tests to clarify the impact of chemical modification on the microbiological activity of either scaffold (Table).     

Immunogenic assessment of plant‐produced human papillomavirus type 16 L1/L2 chimaeras

Cervical cancer is caused by infection with human papillomaviruses (HPV) and is a global concern, particularly in developing countries, which have ~80% of the burden. HPV L1 virus‐like particle (VLP) type–restricted vaccines prevent new infections and associated disease. However, their high cost has limited their application, and cytological screening programmes are still required to detect malignant lesions associated with the nonvaccine types. Thus, there is an urgent need for cheap second‐generation HPV vaccines that protect against multiple types. The objective of this study was to express novel HPV‐16 L1‐based chimaeras, containing cross‐protective epitopes from the L2 minor capsid protein, in tobacco plants. These L1/L2 chimaeras contained epitope sequences derived from HPV‐16 L2 amino acid 108–120, 56–81 or 17–36 substituted into the C‐terminal helix 4 (h4) region of L1 from amino acid 414. All chimaeras were expressed in Nicotiana benthamiana via an Agrobacterium‐mediated transient system and targeted to chloroplasts. The chimaeras were highly expressed with yields of ~1.2 g/kg plant tissue; however, they assembled differently, indicating that the length and nature of the L2 epitope affect VLP assembly. The chimaera containing L2 amino acids 108–120 was the most successful candidate vaccine. It assembled into small VLPs and elicited anti‐L1 and anti‐L2 responses in mice, and antisera neutralized homologous HPV‐16 and heterologous HPV‐52 pseudovirions. The other chimaeras predominantly assembled into capsomeres and other aggregates and elicited weaker humoral immune responses, demonstrating the importance of VLP assembly for the immunogenicity of candidate vaccines.     

Pre‐dawn stomatal opening does not substantially enhance early‐morning photosynthesis in Helianthus annuus

Most C₃ plant species have partially open stomata during the night especially in the 3–5 h before dawn. This pre‐dawn stomatal opening has been hypothesized to enhance early‐morning photosynthesis (A) by reducing diffusion limitations to CO₂ at dawn. We tested this hypothesis in cultivated Helianthus annuus using whole‐shoot gas exchange, leaf level gas exchange and modelling approaches. One hour pre‐dawn low‐humidity treatments were used to reduce pre‐dawn stomatal conductance (g). At the whole‐shoot level, a difference of pre‐dawn g (0.40 versus 0.17 mol m^?2 s^?1) did not significantly affect A during the first hour after dawn. Shorter term effects were investigated with leaf level gas exchange measurements and a difference of pre‐dawn g (0.10 versus 0.04 mol m^?2 s^?1) affected g and A for only 5 min after dawn. The potential effects of a wider range of stomatal apertures were explored with an empirical model of the relationship between A and intercellular CO₂ concentration during the half‐hour after dawn. Modelling results demonstrated that even extremely low pre‐dawn stomatal conductance values have only a minimal effect on early‐morning A for a few minutes after dawn. Thus, we found no evidence that pre‐dawn stomatal opening enhances A.     

Enantiomeric Recognition of Racemic 4‐Aryl‐1,4‐dihydropyridine Derivatives via Chiralpak AD‐H Stationary Phases

The chromatographic chiral resolution of two new series of racemic 4‐substituted‐1,4‐dihydropyridine derivatives was studied on a commercial Chiralpak AD‐H column. Analytes without 5,5‐dimethyl substituents ( 1–15 ) are more efficiently resolved than analytes with 5,5‐dimethyl groups ( 16–30 ). The AD‐H column discriminated between enantiomers through both hydrogen bonding attractions and π–π interactions. This interpretation is in accord with plots of the logarithm of separation factors, log(α), versus σ (Hammett–Swain substituent parameter) and σ⁺ (Brown substituent constant) plots. By elucidating the effects of the remote substituents on these chiral separations, it was shown that the influence of π–π interaction forces increase when steric bulk effects act to decrease the hydrogen bonding attractive forces on the AD‐H column. Chirality 24:854–859, 2012. © 2012 Wiley Periodicals, Inc.     

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)_1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ_1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ_1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ_1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ_1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.