Absolute configuration of an axially chiral sulfonate determined from its optical rotatory dispersion,electronic circular dichroism,and vibrational circular dichroism spectra

The absolute configuration (AC) of an axially chiral sulfonate (aCSO), 3,5‐dimethyl‐2‐(naphthalen‐1‐yl)‐6‐(naphthalen‐1‐yl)benzenesulfonate (labeled as aCSO5), was investigated using optical rotatory dispersion (ORD), electronic circular dichroism (ECD), and vibrational circular dichroism (VCD) spectroscopies. All three methods led to the same conclusion and the AC of aCSO5 is reliably determined to be (−)‐(aR , aR ), or conversely (+)‐(aS , aS ).     

Gender‐dependent regulation of G‐protein‐gated inwardly rectifying potassium current in dorsal raphe neurons in knock‐out mice devoid of the 5‐hydroxytryptamine transporter

Agonists at G‐protein‐coupled receptors in neurons of the dorsal raphe nucleus (DRN) of knock‐out mice devoid of the serotonin transporter (5‐HTT^?/?) exhibit lower efficacy to inhibit cellular discharge than in wild‐type counterparts. Using patch‐clamp whole‐cell recordings, we found that a G‐protein‐gated inwardly rectifying potassium (GIRK) current is involved in the inhibition of spike discharge induced by 5‐HT_1A agonists (5‐carboxamidotryptamine (5‐CT) and (±)‐2‐dipropylamino‐8‐hydroxy‐1,2,3,4‐tetrahydronaphthalene hydrobromide (8‐OH‐DPAT); 50 nM–30 μM) in both wild‐type and 5‐HTT^?/? female and male mice. These effects were mimicked by 5′‐guanylyl‐imido‐diphosphate (Gpp(NH)p; 400 μM) dialysis into cells with differences between genders. The 5‐HTT^?/? knock‐out mutation reduced the current density induced by Gpp(NH)p in females but not in males. These data suggest that the decreased response of 5‐HT_1A receptors to agonists in 5‐HTT^?/? mutants reflects notably alteration in the coupling between G‐proteins and GIRK channels in females but not in males. Accordingly, gender differences in central 5‐HT neurotransmission appear to depend—at least in part—on sex‐related variations in corresponding receptor‐G protein signaling mechanisms. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Tumor‐Targeting Salmonella typhimurium A1‐R Promotes Tumoricidal CD8+ T Cell Tumor Infiltration and Arrests Growth and Metastasis in a Syngeneic Pancreatic‐Cancer Orthotopic Mouse Model

The present study determined the effect of the tumor‐targeting strain Salmonella typhimurium A1‐R (S. typhimurium A1‐R) on CD8⁺ tumor‐infiltrating lymphocytes (TILs) in a syngeneic pancreatic‐cancer orthotopic mouse model. The effect of tumor‐targeting S. typhimurium A1‐R on CD8⁺ TILs was determined on the Pan02 murine pancreatic‐adenocarcinoma implanted orthotopically in the pancreatic tail of C57BL/6 immunocompromised mice. Three weeks after orthotopic implantation, mice were randomized as follows G1: untreated control group (n = 8); and G2: S. typhimurium A1‐R‐treatment group (n = 8, 1 × 10⁷ colony forming units [CFU]/body, iv, weekly, 3 weeks). On the 22nd day from initial treatment, all mice were sacrificed and tumors were harvested. The tumor‐volume ratio was defined as ratio of tumor volume on the 22nd day relative to the 1st day. The tumor volume ratio was significantly lower in the S. typhimurium A1‐R‐treated group (G2) (3.0 ± 2.8) than the untreated control (G1) (39.9 ± 30.7, P < 0.01). Hematoxylin and easin (H&E) staining on tumor sections was performed to evaluate tumor destruction which was classified according to the Evans grading system and found to be much greater in the S. typhimurium A1‐R‐treated mice (G2). Six mice in G1 had peritoneal dissemination, whereas no mice showed peritoneal dissemination in G2 (P < 0.01). Immunohistochemical staining with anti‐mouse CD8⁺ antibody was performed in order to detect TILs determined by calculating the average number of CD8⁺ cells in three high power fields (200×) in the treated and untreated tumors. The TIL score was significantly higher in G2 (133.5 ± 32.2) than G1 (45.1 ± 19.4, P < 0.001). The present study demonstrates that S. typhimurium A1‐R promotes CD8⁺ T cell infiltration and inhibition of tumor growth and metastasis. J. Cell. Biochem. 119: 634–639, 2018. © 2017 Wiley Periodicals, Inc.     

NPY‐Y1 coexpressed with NPY‐Y5 receptors modulate anxiety but not mild social stress response in mice

The Y1 and Y5 receptors for neuropeptide Y have overlapping functions in regulating anxiety. We previously demonstrated that conditional removal of the Y1 receptor in the Y5 receptor expressing neurons in juvenile Npy1r^Y5R?/? mice leads to higher anxiety but no changes in hypothalamus‐pituitary‐adrenocortical axis activity, under basal conditions or after acute restraint stress. In the present study, we used the same conditional system to analyze the specific contribution of limbic neurons coexpressing Y1 and Y5 receptors on the emotional and neuroendocrine responses to social chronic stress, using different housing conditions (isolation vs. group‐housing) as a model. We demonstrated that control Npy1r^2lox male mice housed in groups show increased anxiety and hypothalamus‐pituitary‐adrenocortical axis activity compared with Npy1r^2lox mice isolated for six weeks immediately after weaning. Conversely, Npy1r^Y5R?/? conditional mutants display an anxious‐like behavior but no changes in hypothalamus‐pituitary‐adrenocortical axis activity as compared with their control littermates, independently of housing conditions. These results suggest that group housing constitutes a mild social stress for our B6129S mouse strain and they confirm that the conditional inactivation of Y1 receptors specifically in Y5 receptor containing neurons increases stress‐related anxiety without affecting endocrine stress responses.     

Transient and rapid activation of Akt/GSK‐3β and mTORC1 signaling by D3 dopamine receptor stimulation in dorsal striatum and nucleus accumbens

D2/D3 dopamine receptors (D2R/D3R) agonists regulate Akt, but their effects display a complex time‐course. In addition, the respective roles of D2R and D3R are not defined and downstream targets remain poorly characterized, especially in vivo. These issues were addressed here for D3R. Systemic administration of quinelorane, a D2R/D3R agonist, transiently increased phosphorylation of Akt and GSK‐3β in rat nucleus accumbens and dorsal striatum with maximal effects 10 min after injection. Akt activation was associated with phosphorylation of several effectors of the mammalian target of rapamycin complex 1 (mTORC1): p70S6 kinase, ribosomal protein‐S6 (Ser240/244), and eukaryotic initiation factor‐4E binding protein‐1. The action of quinelorane was antagonized by a D2/D3R antagonist, raclopride, and the selective D3R antagonist S33084, inactive by themselves. Furthermore, no effect of quinerolane was seen in knock‐out mice lacking D3R. In drd1a‐EGFP transgenic mice, quinelorane activated Akt/GSK‐3β in both neurons expressing and lacking D1 receptor. Thus, the stimulation of D3R transiently activates the Akt/GSK‐3β pathway in the two populations of medium‐size spiny neurons of the nucleus accumbens and dorsal striatum. This effect may contribute to the influence of D3R ligands on reward, cognition, and processes disrupted in schizophrenia, drug abuse, and Parkinson's disease.     

Chiral superstructures from homochiral Zn2+, Co2+, Fe2+‐2,6‐bis (aryl ethylimine)pyridine complexes

We report the hierarchical supramolecular organization of metallosupramolecular homochiral complexes 1 ‐Λ‐(S,S,S,S)‐M²⁺/ 1 ‐?‐(R,R,R,R)‐M²⁺ and 2 ‐ Λ‐(S,S,S,S)‐M²⁺/ 2 ‐?‐ (R,R,R,R)‐M²⁺ of M²⁺ = Co²⁺, Fe²⁺, Zn²⁺ metal ions with chiral pseudo‐terpyridine‐type ligands: 1‐ (S,S) or 1‐ (R,R) = 2,6‐bis (naphthyl ethylimine)pyridine and 2‐ (S,S) or 2‐ (R,R) = 2,6‐bis (phenyl‐ethylimine)pyridine. Circular dichroism measurements in solution were used to confirm the enantiomeric nature of all twelve complexes. For crystal structures of 1 ‐ Λ‐ (S,S,S,S)‐M²⁺ or 1 ‐?‐ (R,R,R,R)‐M²⁺ complexes, absolute configurations {? (or P), Λ (or M)} were confirmed by refinement of the Flack parameter x: ?0.007 ≤ x ≤ 0.11 for the single crystals of 1 ‐Λ‐(S,S,S,S)‐M²⁺/ 1 ‐?‐ (R,R,R,R)‐M²⁺, 2 ‐ Λ‐ (S,S,S,S)‐Fe²⁺, and 2 ‐?‐ (R,R,R,R)‐Co²⁺.     

Generation of B6‐Ddx4em1(CreERT2)Utr,a novel CreERT2 knock‐in line,for germ cell lineage by CRISPR/Cas9

Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock‐out mouse studies. In this study, we established a novel knock‐in mouse line, B6‐Ddx4 ^{em1(CreERT2)Utr}, which expresses CreERT2 recombinase under the control of the endogenous DEAD‐box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen‐treated B6‐Ddx4 ^{em1(CreERT2)Utr}::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6‐Ddx4 ^{em1(CreERT2)Utr} is beneficial for studying female and male germ cell development.     

A p57 conditional mutant allele that allows tracking of p57‐expressing cells

p57^Kip2 (p57 ) is a maternally expressed imprinted gene regulating growth arrest which belongs to the CIP/KIP family of cyclin‐dependent kinase inhibitors. While initially identified as a cell cycle arrest protein through inhibition of cyclin and cyclin‐dependent kinase complexes, p57 activity has also been linked to differentiation, apoptosis, and senescence. In addition, p57 has recently been shown to be involved in tumorigenesis and cell fate decisions in stem cells. Yet, p57 function in adult tissues remains poorly characterized due to the perinatal lethality of p57 knock‐out mice. To analyze p57 tissue‐specific activity, we generated a conditional mouse line (p57^FL‐ILZ/+ ) by flanking the coding exons 2–3 by LoxP sites. To track p57‐expressing or mutant cells, the p57^FL‐ILZ allele also contains an IRES‐linked β‐galactosidase reporter inserted in the 3′ UTR of the gene. Here, we show that the β‐galactosidase reporter expression pattern recapitulates p57 tissue specificity during development and in postnatal mice. Furthermore, we crossed the p57^FL‐ILZ/+ mice with PGK‐Cre mice to generate p57^cKO‐ILZ/+ animals with ubiquitous loss of p57. p57^cKO‐ILZ/+ mice display developmental phenotypes analogous to previously described p57 knock‐outs. Thus, p57^FL‐ILZ/+ is a new genetic tool allowing expression and functional conditional analyses of p57.     

Solution structures and backbone dynamics of the ribosomal protein S6 and its permutant P54‐55

The ribosomal protein S6 from Thermus thermophilus has served as a model system for the study of protein folding, especially for understanding the effects of circular permutations of secondary structure elements. This study presents the structure of a permutant protein, the 96‐residue P^54‐55, and the structure of its 101‐residue parent protein S6^wt in solution. The data also characterizes the effects of circular permutation on the backbone dynamics of S6. Consistent with crystallographic data on S6^wt, the overall solution structures of both P^54‐55 and S6^wt show a β‐sheet of four antiparallel β‐strands with two α‐helices packed on one side of the sheet. In clear contrast to the crystal data, however, the solution structure of S6^wt reveals a disordered loop in the region between β‐strands 2 and 3 (Leu43‐Phe60) instead of a well‐ordered stretch and associated hydrophobic mini‐core observed in the crystal structure. Moreover, the data for P^54‐55 show that the joined wild‐type N‐ and C‐terminals form a dynamically robust stretch with a hairpin structure that complies with the in silico design. Taken together, the results explain why the loop region of the S6^wt structure is relatively insensitive to mutational perturbations, and why P^54‐55 is more stable than S6^wt: the permutant incision at Lys54‐Asp55 is energetically neutral by being located in an already disordered loop whereas the new hairpin between the wild‐type N‐ and C‐termini is stabilizing.     

A novel role for the mitochondrial HTRA2/OMI protease in aging

HTRA2/OMI is an ATP-independent serine protease located in the intermembrane space of the mitochondria and is thought to function as a protein quality control protease. Our previous studies showed that loss of the enzymatic activity of HTRA2 due to a Ser276Cys missense mutation in its catalytic domain is associated with early onset neurodegeneration, multiple tissue atrophy and premature lethality in homozygous htra2^mnd2 mice, suggesting that HTRA2 is neuroprotective. To further investigate the role of HTRA2 in neuronal cell survival and the impact of its loss of function in non-neuronal tissues of adult mice, we generated transgenic htra2^mnd2 mice expressing a neuron-targeted human HTRA2 transgene. Notably, this HTRA2 transgene rescues htra2^mnd2 mice from early onset neurodegeneration, and other phenotypic abnormalities and prevents their early death, indicating that HTRA2 activity in neuronal mitochondria is important for neuronal cell survival. However, as the rescued htra2^mnd2 mice grow older they exhibit specific phenotypic abnormalities indicative of premature aging. These include premature weight loss, osteoporosis, lordokyphosis, muscle atrophy, heart enlargement, increased autophagy and reduced life span. There is also a significant increase in the levels of clonally expanded mitochondrial DNA (mtDNA) deletions in their tissues. Our findings suggest that HTRA2-regulated protein quality control in the intermembrane space of mitochondria is important for the maintenance of mitochondrial homeostasis, and loss of HTRA2 activity can lead to both neurodegeneration and aging.     

Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene

CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock‐out mice. This represents creation of the first gene knock‐out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock‐in (Clic1^FN) allele, followed by Clic1 knock‐out (Clic1^−/−) mice by crossing Clic1^FN allele with TNAP‐cre mice, resulting in germline gene deletion through Cre‐mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1^−/− mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y₁₂ receptor signaling. genesis 48:127–136, 2010. © 2010 Wiley‐Liss, Inc.     

Design of experiment assisted concurrent enantioseparation of bupropion and hydroxybupropion by high‐performance thin‐layer chromatography

A simple and efficient high‐performance thin‐layer chromatographic method was developed for chiral separation of rac ‐bupropion (BUP) and its active metabolite rac ‐hydroxybupropion (HBUP). Design of experiment (DoE)‐based optimization was adopted instead of a conventional trial‐and‐error approach. The Box–Behnken design surface response model was used and the operating variables were optimized based on 17 trials design. The optimized method involved impregnation of chiral reagent, L(+)‐tartaric acid, in the stationary phase with simultaneous addition in the mobile phase, which consisted of acetonitrile : methanol : dichloromethane : 0.50% L‐tartaric acid (6.75:1.0:1.0:0.25, v /v /v /v ). Under the optimized conditions, the resolution factor between the enantiomers of BUP and HBUP was 6.30 and 9.26, respectively. The limit of detection and limit of quantitation for (R)‐BUP, (S)‐BUP, (R,R)‐HBUP, and (S,S)‐HBUP were 9.23 and 30.78 ng spot⁻¹, 10.32 and 34.40 ng spot⁻¹, 12.19 and 40.65 ng spot⁻¹, and 14.26 and 47.53 ng spot⁻¹, respectively. The interaction of L‐tartaric acid with analytes and their retention behavior was thermodynamically investigated using van't Hoff's plots. The developed method was validated as per the International Conference on Harmonization guidelines. Finally, the method was successfully applied to resolve and quantify the enantiomeric content from marketed tablets as well as spiked plasma samples.     

Embryonic expression of an Nkx2‐5/Cre gene using ROSA26 reporter mice

Summary: Nkx2‐5, one of the earliest cardiac‐specific markers in vertebrate embryos, was used as a genetic locus to knock in the Cre recombinase gene by homologous recombination. Offspring resulting from heterozygous Nkx2‐5/Cre mice mated to ROSA26 (R26R) reporter mice provided a model system for following Nkx2‐5 gene activity by β‐galactosidase (β‐gal) activity. β‐gal activity was initially observed in the early cardiac crescent, cardiomyocytes of the looping heart tube, and in the epithelium of the first pharyngeal arch. In later stage embryos (10.5–13.5 days postcoitum, dpc), β‐gal activity was observed in the stomach and spleen, the dorsum of the tongue, and in the condensing primordium of the tooth. The Nkx2‐5/Cre mouse model should provide a useful genetic resource to elucidate the role of loxP manipulated genetic targets in cardiogenesis and other developmental processes. genesis 31:176–180, 2001. © 2001 Wiley‐Liss, Inc.     

The cardiac repair benefits of inflammation do not persist: evidence from mast cell implantation

Multiple mechanisms contribute to progressive cardiac dysfunction after myocardial infarction (MI) and inflammation is an important mediator. Mast cells (MCs) trigger inflammation after MI by releasing bio‐active factors that contribute to healing. c‐Kit‐deficient (Kit^W/W‐v) mice have dysfunctional MCs and develop severe ventricular dilatation post‐MI. We explored the role of MCs in post‐MI repair. Mouse wild‐type (WT) and Kit^W/W‐v MCs were obtained from bone marrow (BM). MC effects on fibroblasts were examined in vitro by proliferation and gel contraction assays. MCs were implanted into infarcted mouse hearts and their effects were evaluated using molecular, cellular and cardiac functional analyses. In contrast to WT, Kit^W/W‐v MC transplantation into Kit^W/W‐v mice did not improve cardiac function or scar size post‐MI. Kit^W/W‐v MCs induced significantly reduced fibroblast proliferation and contraction compared to WT MCs. MC influence on fibroblast proliferation was Basic fibroblast growth factor (bFGF)‐dependent and MC‐induced fibroblast contractility functioned through transforming growth factor (TGF)‐β. WT MCs transiently rescue cardiac function early post‐MI, but the benefits of BM cell implantation lasted longer. MCs induced increased inflammation compared to the BM‐injected mice, with increased neutrophil infiltration and infarct tumour necrosis factor‐α (TNF‐α) concentration. This augmented inflammation was followed by increased angiogenesis and myofibroblast formation and reduced scar size at early time‐points. Similar to the functional data, these beneficial effects were transient, largely vanishing by day 28. Dysfunctional Kit^W/W‐v MCs were unable to rescue cardiac function post‐MI. WT MC implantation transiently enhanced angiogenesis and cardiac function. These data suggest that increased inflammation is beneficial to cardiac repair, but these effects are not persistent.     

Analysis of Oxcarbazepine and the 10‐Hydroxycarbazepine Enantiomers in Plasma by LC‐MS/MS: Application in a Pharmacokinetic Study

Oxcarbazepine is a second‐generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic–clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10‐hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)‐(+)‐ and R‐(?)‐MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Aliquots of 100 μL of plasma were extracted with a mixture of methyl tert‐butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD‐H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC‐MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S‐(+)‐MHD enantiomer compared to R‐(?)‐MHD and an AUC^0‐12 _{S‐(+)/R‐(?)} ratio of 5.44. Chirality 25:897–903, 2013. © 2013 Wiley Periodicals, Inc.     

\Cytokinin oxidase/dehydrogenase (CKX) activity in wild and ethylene-insensitive mutant eti5 type of Arabidopsis thaliana (L.) Heynh plants and the effect of cytokinin N1-(2-chloro-4-pyridyl)-N2-phenylurea on enzymatic activity and leaf morphology

The specific activity of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) (CKX) was determined in leaves of wild type (wt) and ethylene-insensitive mutant (eti5) of Arabidopsis thaliana (L.) Heynh plants. Comparative studies showed that this mutation has lower basal CKX activity than wt. Application of 4PU-30 (N¹-(2-chloro-4-pyridyl)-N²-phenylurea) resulted in decreased CKX activity in both wt and mutant plants. The treatment increased leaf blade thickness and the volume of chlorophyll-containing cells per unit leaf area in wt but these changes were not observed in the eti5 mutant. The reduction in chlorophyll “a” and “b”, as well as in carotenoids content in the treated wt tissues resulting from altered leaf morphology was not detected in eti5 plants.