Preparation of a molecularly imprinted polymer for the solid-phase extraction of scopolamine with hyoscyamine as a dummy template molecule

Molecularly imprinted polymers (MIPs) selective for scopolamine were produced using hyoscyamine (a close structural analogue) as template molecule. The produced polymers were used as media for solid-phase extraction, exhibiting selective binding properties for the analyte from biological samples. Human and calf urine and serum were processed on the MIP under various extraction protocols. The best performance was observed after loading the analyte in aqueous environment facilitating retention on the MIP by non-selective hydrophobic interactions. The MIPs were subsequently washed using an optimised solvent system to enable selective desorption of the analyte. Other related and non-related compounds were accessed to evaluate molecular recognition properties. Recoveries of up to 79% were achieved for the analyte of interest from biological samples.

A Kinetic-Based Model of Radiation-Induced Intercellular Signalling

It is now widely accepted that intercellular communication can cause significant variations in cellular responses to genotoxic stress. The radiation-induced bystander effect is a prime example of this effect, where cells shielded from radiation exposure see a significant reduction in survival when cultured with irradiated cells. However, there is a lack of robust, quantitative models of this effect which are widely applicable. In this work, we present a novel mathematical model of radiation-induced intercellular signalling which incorporates signal production and response kinetics together with the effects of direct irradiation, and test it against published data sets, including modulated field exposures. This model suggests that these so-called "bystander" effects play a significant role in determining cellular survival, even in directly irradiated populations, meaning that the inclusion of intercellular communication may be essential to produce robust models of radio-biological outcomes in clinically relevant in vivo situations.

Host cell kinases, α5 and β1 integrins, and Rac1 signalling on the microtubule cytoskeleton are important for non-typable Haemophilus influenzae invasion of respiratory epithelial cells

Non-typable Haemophilus influenzae (NTHi) is a common commensal of the human nasopharynx, but causes opportunistic infection when the respiratory tract is compromised by infection or disease. The ability of NTHi to invade epithelial cells has been described, but the underlying molecular mechanisms are poorly characterized. We previously determined that NTHi promotes phosphorylation of the serine-threonine kinase Akt in A549 human lung epithelial cells, and that Akt phosphorylation and NTHi cell invasion are prevented by inhibition of phosphoinositide 3-kinase (PI3K). Because PI3K-Akt signalling is associated with several host cell networks, the purpose of the current study was to identify eukaryotic molecules important for NTHi epithelial invasion. We found that inhibition of Akt activity reduced NTHi internalization; differently, bacterial entry was increased by phospholipase C?1 inhibition but was not affected by protein kinase inhibition. We also found that a5 and ß1 integrins, and the tyrosine kinases focal adhesion kinase and Src, are important for NTHi A549 cell invasion. NTHi internalization was shown to be favoured by activation of Rac1 guanosine triphosphatase (GTPase), together with the guanine nucleotide exchange factor Vav2 and the effector Pak1. Also, Pak1 might be associated with inactivation of the microtubule destabilizing agent Op18/stathmin, to facilitate microtubule polymerization and NTHi entry. Conversely, inhibition of RhoA GTPase and its effector ROCK increased the number of internalized bacteria. Src and Rac1 were found to be important for NTHi-triggered Akt phosphorylation. An increase in host cyclic AMP reduced bacterial entry, which was linked to protein kinase A. These findings suggest that NTHi finely manipulates host signalling molecules to invade respiratory epithelial cells.

Changed reactivity of the 1-bromo-4-nitrobenzene radical anion in a room temperature ionic liquid

Radical anions of 1-bromo-4-nitrobenzene (p-BrC6H4NO2) are shown to be reactive in the room temperature ionic liquid N-butyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide, ([C(4)mPyrr][NTf2]), by means of voltammetric measurements. In particular, they are shown to react via a DISP type mechanism such that the electrolysis of p-BrC6H4NO2 occurs consuming between one and two electrons per reactant molecule, leading to the formation of the nitrobenzene radical anion and bromide ions. This behaviour is a stark contrast to that in conventional non-aqueous solvents such as acetonitrile, dimethyl sulfoxide or N,N-dimethylformamide, which suggests that the ionic solvent promotes the reactivity of the radical anion, probably via stabilisation of the charged products.

Proteomic analysis of colorectal cancer metastasis:stathmin-1 revealed as a player in cancer cell migration and prognostic marker

Metastasis accounts largely for the high mortality rate of colorectal cancer (CRC) patients. In this study, we performed comparative proteome analysis of primary CRC cell lines HCT-116 and its metastatic derivative E1 using 2-D DIGE. We identified 74 differentially expressed proteins, many of which function in transcription, translation, angiogenesis signal transduction, or cytoskeletal remodeling pathways, which are indispensable cellular processes involved in the metastatic cascade. Among these proteins, stathmin-1 (STMN1) was found to be highly up-regulated in E1 as compared to HCT-116 and was thus selected for further functional studies. Our results showed that perturbations in STMN1 levels resulted in significant changes in cell migration, invasion, adhesion, and colony formation. We further showed that the differential expression of STMN1 correlated with the cells' metastatic potential in other paradigms of CRC models. Using immunohistochemistry, we also showed that STMN1 was highly expressed in colorectal primary tumors and metastatic tissues as compared to the adjacent normal colorectal tissues. Furthermore, we also showed via tissue microarray analyses of 324 CRC tissues and Kaplan-Meier survival plot that CRC patients with higher expression of STMN1 have poorer prognosis.

Serum Inflammatory Markers and Preeclampsia in Type 1 Diabetes:A prospective study

OBJECTIVE Inflammation and endothelial dysfunction have been associated with the immunobiology of preeclampsia (PE), a significant cause of adverse pregnancy outcomes. The prevalence of PE is elevated several fold in the presence of maternal type 1 diabetes mellitus (T1DM). Although cross-sectional studies of pregnancies among women without diabetes have shown altered inflammatory markers in the presence of PE, longitudinal studies of diabetic women are lacking. In maternal serum samples, we examined the temporal associations of markers of inflammation with the subsequent development of PE in women with T1DM. RESEARCH DESIGN AND METHODS We conducted longitudinal analyses of serum C-reactive protein (CRP), adhesion molecules, and cytokines during the first (mean ± SD, 12.2 ± 1.9 weeks), second (21.6 ± 1.5 weeks), and third (31.5 ± 1.7 weeks) trimesters of pregnancy (visits 1-3, respectively). All study visits took place before the onset of PE. Covariates were BMI, HbA1c, age of onset, duration of diabetes, and mean arterial pressure. RESULTS In women with T1DM who developed PE versus those who remained normotensive, CRP tended to be higher at visits 1 (P = 0.07) and 2 (P = 0.06) and was significantly higher at visit 3 (P <0.05); soluble E-selectin and interferon-?-inducible protein-10 (IP-10) were significantly higher at visit 3; interleukin-1 receptor antagonist (IL-1ra) and eotaxin were higher and lower, respectively, at visit 2 (all P <0.05). These conclusions persisted following adjustment for covariates. CONCLUSIONS In pregnant women with T1DM, elevated CRP, soluble E-selectin, IL-1ra, and IP-10 and lower eotaxin were associated with subsequent PE. The role of inflammatory factors as markers and potential mechanisms of the high prevalence of PE in T1DM merits further investigation.

The role of SRC-CAS interactions in cellular transformation:ectopic expression of the carboxy terminus of CAS inhibits SRC-CAS interaction but has no effect on cellular transformation

Several lines of evidence indicate that the adapter molecule p130CAS (crk-associated substrate (CAS)) is required for src-mediated cellular transformation. CAS has been shown to be heavily tyrosine-phosphorylated in src-transformed cells, and genetic variants of src that are deficient in CAS binding are also unable to mediate cellular transformation. In this report, we investigated whether CAS phosphorylation and/or its association with src are required elements of the transformation process. Expression of the carboxy-terminal src binding domain of CAS in Rat 1 fibroblasts expressing a temperature-sensitive allele of v-src inhibited the formation of src-CAS complexes and also inhibited tyrosine phosphorylation of CAS. However, expression of this protein had no effect on morphological transformation, src-mediated actin rearrangements, or anchorage-independent growth of these cells when grown at the src-permissive temperature. Thus, the ability of activated src to mediate cellular transformation is either largely independent of endogenous CAS phosphorylation and/or its association with CAS or, alternatively, the carboxy-terminus of CAS may substitute for endogenous CAS in the process of src-mediated transformation.

Regulation of apoptotic protease activating factor-1 oligomerization and apoptosis by the WD-40 repeat region

Apoptotic protease activating factor-1 (Apaf-1) has been identified as a proximal activator of caspase-9 in cell death pathways that trigger mitochondrial damage and cytochrome c release. The mechanism of Apaf-1 action is unclear but has been proposed to involve the clustering of caspase-9 molecules, thereby facilitating autoprocessing of adjacent zymogens. Here we show that Apaf-1 can dimerize via the CED-4 homologous and linker domains of the molecule providing a means by which Apaf-1 can promote the clustering of caspase-9 and facilitate its activation. Apaf-1 dimerization was repressed by the C-terminal half of the molecule, which contains multiple WD-40 repeats, but this repression was overcome in the presence of cytochrome c and dATP. Removal of the WD-40 repeat region resulted in a constitutively active Apaf-1 that exhibited greater cytotoxicity in transient transfection assays when compared with full-length Apaf-1. These data suggest a mechanism for Apaf-1 function and reveal an important regulatory role for the WD-40 repeat region.

Cathelicidin LL-37:a defense molecule with a potential role in psoriasis pathogenesis

Epidermal keratinocytes produce and secrete antimicrobial peptides (AMPs) that subsequently form a chemical shield on the skin surface. Cathelicidins are one family of AMPs in skin with various further immune functions. Consequently, dysfunction of these peptides has been implicated in the pathogenesis of inflammatory skin disease. In particular, the cathelicidin LL-37 is overexpressed in inflamed skin in psoriasis, binds to extracellular self-DNA released from dying cells and converts self-DNA in a potent stimulus for plasmacytoid dendritic cells (pDCs). Subsequently, pDCs secrete type I interferons and trigger an auto-inflammatory cascade. Paradoxically, therapies targeting the vitamin D pathway such as vitamin D analogues or UVB phototherapy ameliorate cutaneous inflammation in psoriasis but strongly induce cathelicidin expression in skin at the same time. Current evidence now suggests that self-DNA present in the cytosol of keratinocytes is also pro-inflammatory active and triggers IL-1β secretion in psoriatic lesions through the AIM2 inflammasome. This time, however, binding of LL-37 to self-DNA neutralizes DNA-mediated inflammation. Hence, cathelicidin LL-37 shows contrasting roles in skin inflammation in psoriasis and might serve as a target for novel therapies for this chronic skin disease.

Controlling Surface Topology and Functionality of Electrospun Fibers on the Nanoscale using Amphiphilic Block Copolymers To Direct Mesenchymal Progenitor Cell Adhesion

Surface patterning in three dimensions is of great importance in biomaterials design for controlling cell behavior. A facile one-step functionalization of biodegradable PDLLA fibers using amphiphilic diblock copolymers is demonstrated here to systematically vary the fiber surface composition. The copolymers comprise a hydrophilic poly[oligo(ethylene glycol) methacrylate] (POEGMA), poly[(2-methacryloyloxy)ethyl phosphorylcholine] (PMPC), or poly[2-(dimethylamino)ethyl methacrylate)] (PDMAEMA) block and a hydrophobic poly(l-lactide) (PLA) block. The block copolymer-modified fibers have increased surface hydrophilicity compared to that of PDLLA fibers. Mixtures of PLAPMPC and PLAPOEGMA copolymers are utilized to exploit microphase separation of the incompatible hydrophilic PMPC and POEGMA blocks at the fiber surface. Conjugation of an RGD cell-adhesive peptide to one hydrophilic block (POEGMA) using thiol-ene chemistry produces fibers with domains of cell-adhesive (POEGMA) and cell-inert (PMPC) sites, mimicking the adhesive properties of the extracellular matrix (ECM). Human mesenchymal progenitor cells (hES-MPs) showed much better adhesion to the fibers with surface-adhesive heterogeneity compared to that to fibers with only adhesive or only inert surface chemistries.

The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases

UNLABELLED: Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins.

IMPORTANCE: It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by the analysis of several crystal structures, but no information has been available to date for the unrelated HD-GYP class of hydrolases. Here we present the multidomain structure of an unusual member of the HD-GYP family from the predatory bacterium Bdellovibrio bacteriovorus and detail the features that distinguish it from the wider structural family of general HD fold hydrolases. The structure reveals how a binuclear iron center is formed from several conserved residues and provides a basis for understanding HD-GYP family sequence requirements for c-di-GMP hydrolysis.

On the halogenation of N(1),N(2)-di-t-Boc-5-hydroxy-piperazic acid esters and the conformational preferences of their 5-halo-piperazic acid products. The importance of A1,3 rotameric-strain in determining N(2)-acyl piperazic acid ring conformation

In this Letter, an unambiguous synthetic strategy is reported for the preparation of enantiomerically purecis-5-halo-piperazic acid derivatives in single diastereoisomer form. Contrary to the recent report by Shin and co-workers (Chem. Lett. 2001, 1172), in which it is claimed that the Ph₃P and N-chlorosuccinimide (NCS)-mediated chlorination of (3R,5S)-trans-N(1),N(2)-di-t-Boc-5-hydroxy-piperazic acid derivative 1proceeds with retention of configuration at C(5) to give 2, we now show that this and related Ph₃P-mediated halogenations all occur with S_N2 inversion at the alcohol center, as is customary for such reactions. Specifically, we demonstrate that the (3R,5S)-trans-5-Cl-piperazic acid derivative 2 claimed by Shin and co-workers (Chem. Lett. 2001, 1172) is in actual fact the chlorinated (3S,5R)-enantiomer 6, which must have been prepared from the cis-(3S,5S)-alcohol 3, a molecule whose synthesis is not formally described in the Shin paper. We further show here that the cis-(3R,5R)-5-Cl-Piz 13 claimed by Shin and co-workers inChem. Lett. 2001, 1172, is also (3S,5R)-trans-5-Cl-Piz 6. Authentic 13 has now been synthesized by us, for the very first time, here. Since Lindsley and Kennedy have recently utilized the now invalid Shin and co-workers’ retentive Ph₃P/NCS chlorination procedure on 1 in their synthetic approach to piperazimycin A (Tetrahedron Lett. 2010, 51, 2493), it follows that their claimed 5-Cl-Piz-containing dipeptide 25 probably has the alternate structure 26, where the 5-Cl-Piz residue has a 3,5-cis-configuration. The aforementioned stereochemical misassignments appear to have come from a mix-up of starting materials by Shin and co-workers (Chem. Lett. 2001, 1172), and an under-appreciation of the various steric and conformational effects that operate in N(2)-acylated piperazic acid systems, most especially rotameric A^1,3-strain. The latter has now been unambiguously delineated and defined here under the banner of the A^1,3-rotamer effect.

Heme oxygenase-1 derived carbon monoxide permits maturation of myeloid cells

Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1(flfl)), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation.

The histamine H4 receptor is a potent inhibitor of adhesion-dependent degranulation in human neutrophils

The histamine H4 receptor regulates the inflammatory response. However, it is not known whether this receptor has a functional role in human neutrophils. We found that fMLP (1 μM), but not histamine (0.1-1 μM), induced Mac-1-dependent adhesion, polarization, and degranulation (release of lactoferrin). A pretreatment of neutrophils with histamine (0.001-1 μM) or JNJ 28610244 (0.1-10 μM), a specific H4 receptor agonist, led to inhibition of degranulation. Total inhibition of degranulation was obtained with 0.1 μM histamine and 10 μM JNJ 28610244. Furthermore, such inhibition by histamine of degranulation was reversed by JNJ 7777120 and JNJ 28307474, two selective H4 receptor antagonists. However, neither histamine nor the H4 receptor agonist JNJ 28610244 prevented fMLP-induced, Mac-1-dependent adhesion, indicating that the H4 receptor may block signals emanating from Mac-1-controlling degranulation. Likewise, engagement of the H4 receptor by the selective agonist JNJ 28610244 blocked Mac-1-dependent activation of p38 MAPK, the kinase that controls neutrophil degranulation. We also show expression of the H4 receptor at the mRNA level in ultrapure human neutrophils and myeloid leukemia PLB-985 cells. We concluded that engagement of this receptor by selective H4 receptor agonists may represent a good, therapeutic approach to accelerate resolution of inflammation.

CD44-Mediated Activaton of α5β1-Intergrin, Cortactin and Paxillin Signaling Underpins Ashesion of basal-like breat cancer cells to Endotherlium and Fibronectin- enriched Matrices

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanisms. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche.