Polypeptide chains containing D-gamma-hydroxyvaline

Life has an unexplained and distinct l-homochirality. Proteins typically incorporate only l-amino acids into their sequences. In the present study, d-Val and d-gamma-hydroxyvaline (d-Hyv; V) have been found within ribosomally expressed polypeptide chains. Four conopeptides were initially isolated, gld-V/gld-V'from the venom of Conus gladiator and mus-V/mus-V' from the venom of Conus mus. Their complete sequences (gld-V/gld-V' = Ala-Hyp-Ala-Asn-Ser-d-Hyv-Trp-Ser and mus-V/mus-V' = Ser-Hyp-Ala-Asn-Ser-d-Hyv-Trp-Ser) were determined by a combination of nano/pico-NMR and MS/MS methods. The amino acid triad that contains the gamma-hydroxylated residue, Ser-d-Hyv-Trp, is a novel structural motif that is stabilized by specific interactions between the d-amino acid and its neighboring l-counterparts. These interactions inhibit lactonization, a peptide backbone scission process that would normally be initiated by gamma-hydroxylated residues. Conopeptides possessing the Ser-d-Hyv-Trp motif have been termed gamma-hydroxyconophans. We have also isolated analogous conopeptides (gld-V and mus-V) containing d-Val instead of d-Hyv; these are termed conophans. gamma-Hydroxyconophans and conophans are particularly atypical because (i) they are not constrained as most conopeptides, (ii) they are extremely short in length, (iii) they have a high content of hydroxylated residues, and (iv) their sequences have no close match with other peptides in sequence databases. Their modifications appear to be part of a novel hyperhydroxylation mechanism found within the venom of cone snails that enhances neuronal targeting. The finding of d-Val and d-Hyv within this family of peptides suggests the existence of a corresponding d-stereospecific enzyme capable of d-Val oxidation.     

Novel cysteine proteinase inhibitors homologous to the proregions of cysteine proteinases

Propeptides of papain-like cysteine proteinases such as papain, cathepsins B, L and S are potent inhibitors of their cognate cysteine proteinases with Ki values in the nanomolar range, and they exhibit highest inhibition selectivity for enzymes from which they originate. Recent studies have identified novel inhibitor proteins that are homologous to the proregions of papain-like cysteine proteinases. Mouse activated T-lymphocytes express cytotoxic T-lymphocyte antigen (CTLA-2), which is homologous to the proregion of mouse cathepsin L. CTLA-2 exhibits inhibitory activities to several cysteine proteinases. We have also identified a similar propeptide-like cysteine proteinase inhibitor, Bombyx cysteine proteinase inhibitor (BCPI), in the silkmoth Bombyx mori. BCPI is a slow and tight binding inhibitor of cathepsin L-like cysteine proteinases with Ki values in picomolar range, and the inhibition is highly selective towards these proteinases just like the propeptides. Recent genome analyses have shown the expression of similar propeptide-like proteins in Drosophila and rat, suggesting the presence of a novel class of cysteine proteinase inhibitors in a variety of organisms. Studies of the gene structures and phylogenetic analysis have shown that genes of the propeptide-like cysteine proteinase inhibitors have emerged from ancestor genes of their parental enzymes.     

Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates

Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying protein aggregation diseases. Here we report the chemical design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can be utilized as ligands for detection of protein aggregates. Certain molecular requirements were shown to be necessary for detecting (i) early non-thioflavinophilic protein assemblies of Aβ1-42 and insulin preceding the formation of amyloid fibrils and (ii) for obtaining distinct spectral signatures of the two main pathological hallmarks observed in human Alzheimer's diease brain tissue (Aβ plaques and neurofibrillary tangles). Our findings suggest that a superior anionic LCO-based ligand should have a backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated thiophene backbone. Such LCOs will be highly useful for studying the underlying molecular events of protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for these diseases.     

Biotransformation of Valdecoxib by Plant Cell Cultures

Valdecoxib is a new anti-inflammatory drug that is highly selective for inhibition of the inducible form of cyclooxygenase (COX-2). In the present study, biotransformation of valdecoxib was investigated in cell cultures of five medicinal plants, viz., Catharanthus roseus, Azadirachta indica, Capsicum annuum, Ervatamia heyneana, and Nicotiana tabacum. Identification of the biotransformed products was carried out by using high-performance liquid chromatography coupled with diode array detection and liquid chromatography–tandem mass spectrometry analysis. All the cultures transformed valdecoxib into more polar compounds, and C. roseus also produced one unknown compound that is less polar than the substrate. The reactions performed by these plant cell cultures include hydroxylation, methylation, and demethylation. Optimization studies were performed to investigate the effect of the day of extraction and substrate concentration on biotransformation.     

Recent advances in clinical microdialysis

Clinical microdialysis (MD) is a minimally invasive sampling technique that offers selective in-vivo measurement of free, active drug or biomolecule concentrations in human tissues and organs. From a regulatory perspective, MD can thus be seen as a suitable scientific tool that meets regulatory requirements for the study of tissue distribution or bioequivalence during drug development. From a clinical perspective, the use of MD in different applications has shown the potential to rationalize drug-dosing regimens and to influence clinical decision-making, although validation and correlation of MD-derived results with clinical response are required to promote routine clinical use of the technique. From an analytical perspective, highly sensitive analytical systems have increasingly become available for MD-sample analysis, and these have further improved the quality and the power of MD-derived information. Given the constant development in recent years, MD data might become an important part of new drug submissions and clinical treatment algorithms, and might positively influence patient benefit in the future.     

Metronomic Photodynamic Therapy as a New Paradigm for Photodynamic Therapy: Rationale and Preclinical Evaluation of Technical Feasibility for Treating Malignant Brain Tumors¶

The concept of metronomic photodynamic therapy (mPDT) is presented, in which both the photosensitizer and light are delivered continuously at low rates for extended periods of time to increase selective tumor cell kill through apoptosis. The focus of the present preclinical study is on mPDT treatment of malignant brain tumors, in which selectivity tumor cell killing versus damage to normal brain is critical. Previous studies have shown that low‐dose PDT using 5‐aminolevulinic acid (ALA)‐induced protoporphyrin IX(PpIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue or apoptosis in the latter. On the basis of the levels of apoptosis achieved and model calculations of brain tumor growth rates, metronomic delivery or multiple PDT treatments, such as hyperfractionation, are likely required to produce enough tumor cell kill to be an effective therapy. In vitro studies confirm that ALA‐mPDT induces a higher incidence of apoptotic (terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate, sodium salt nick‐end labeling positive) cells as compared with an acute, high‐dose regimen (ALA‐αPDT). In vivo, mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of devices for extended light delivery while allowing unencumbered movement. In rat models, ALA administration via the drinking water has been accomplished at very high doses (up to 10 times therapeutic dose) for up to 10 days, and ex vivo spectro‐fluorimetry of tumor (9L gliosarcoma) and normal brain demonstrates a 3–4 fold increase in the tumor‐to‐brain ratio of PpIX concentration, without evidence of toxicity. After mPDT treatment, histological staining reveals extensive apoptosis within the tumor periphery and surrounding microinvading colonies that is not evident in normal brain or tumor before treatment. Prototype light sources and delivery devices were found to be practical, either using a laser diode or light‐emitting diode (LED) coupled to an implanted optical fiber in the rat model or a directly implanted LED using a rabbit model. The combined delivery of both drug and light during an extended period, without compromising survival of the animals, is demonstrated. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.     

Conotoxins and structural biology: a prospective paradigm for drug discovery

Understanding the interactions between activating or antagonizing ligands and their cognate receptors at a molecular level offers promise for the development of pharmacological therapeutics for CNS disorders. The discovery of novel molecules that are capable of discriminating between the varied molecular subunits or isoforms of ion channels should provide a more detailed understanding of the pathophysiology of many CNS disorders. Abundant natural sources of pharmacologically active agents that demonstrate this refined selectivity and specificity are found in the animal toxins of venomous species including: snakes, spiders and the marine snail of the genus Conus. The uniquely fascinating combinatorial ability of the marine snail, genus Conus to modify the pharmacological properties of these neurotoxins or conopeptides within its venom is depicted throughout this review. The myriad of posttranslational modifications and disulfide bonded architectures that have been identified in the conopeptides, are described with an emphasis on the unique pharmacological properties and receptor target specificities that have been ascribed to each of these modifications. The ability of NMR spectroscopy to provide three-dimensional structural information within the interaction interface for both the ligand and target protein following complex formation and its application to conopeptide drug discovery are discussed. Similarly, the strength of merging NMR spectroscopy data with ab initio "restrained soft-docking" for rational pharmacophore design and the identification of lead compounds from in silico library screens will also be discussed. The initial phases of this stratagem are illustrated using two toxin antagonists and the recently determined structure of the KcsA potassium channel. These data exemplify the utility of this approach in elucidating important molecular interfaces of specific toxin-receptor/ion channel complexes, which can be further exploited in drug discovery initiatives.     

LC with Novel Electrochemical Detection for Analysis of Monoamine Neurotransmitters in Rat Brain After Administration of (R)-Salsolinol and (R)-N-Methylsalsolinol

A novel electrochemical detector with an acetylcholine film has been developed for HPLC. The chemically modified electrode is an efficient electrocatalyst of (R)-salsolinol ((R)-Sal), (R)-N-methylsalsolinol ((R)-NMSal), and monoamine neurotransmitters, enabling highly sensitive detection. The electrode is also stable and long-lived. Combined with microdialysis sampling, HPLC with the novel detector enabled successful study of changes in the concentrations of monoamine neurotransmitters in rat brain after the injection of (R)-salsolinol and (R)-N-methylsalsolinol.     

Carbohydrate arrays for functional studies of carbohydrates

Carbohydrates, as components of glycoproteins, glycolipids and proteoglycans, play an important biological role as recognition markers through carbohydrate-protein interactions. For the most part, biophysical and biochemical methods have been used to analyze these biomolecular interactions. In contrast, less attention has been given to the development of high-throughput procedures to elucidate carbohydrate-protein recognition events. Recently, carbohydrate arrays were developed and employed as a novel high-throughput analytic tool for monitoring carbohydrate-protein interactions. This technique has been used to profile protein binding and enzymatic activity. The results have shown that carbohydrate binding to the corresponding lectins is highly selective and that the relative binding affinities are well correlated with those obtained from solution-based assays. In addition, this effort demonstrated that carbohydrate arrays could be also utilized to identify and characterize novel carbohydrate-binding proteins or carbohydrate-processing enzymes. Finally, the results of this investigation showed that lectin-carbohydrate binding affinities could be quantitatively assessed by determining IC50 values for soluble carbohydrates with the carbohydrate arrays. The results of these studies suggest that carbohydrate arrays have the potential of playing an important role in basic researches, the diagnoses of diseases and drug discovery.     

The complex dielectric spectrum of heart tissue during ischemia

INTRODUCTION: Because of the variety of tissue structures, the interpretation of the passive complex dielectric permittivity spectrum epsilon (omega) of the heart is still a problem. The aim of this work was to correlate epsilon (omega) of heart tissue with physical processes on cellular level. METHODS: epsilon (omega) of canine hearts was continuously measured in the range from 10 Hz to 400 MHz during cardioplegic perfusion and during following ischemia. Cardioplegic perfusion was performed with HTK (Custodiol) without or with heptanol, in order to produce electrical cell uncoupling via the closure of gap junctions. To analyse epsilon (omega), we present two heart models which consider cell shape, electrical cell coupling, and dielectric polarisation of cell membranes and membranes of intracellular structures. RESULTS: epsilon (omega) of heart tissue shows an alpha-, beta-, and gamma-dispersion. epsilon (omega) remains unchanged during cardioplegic perfusion with HTK, but if heptanol is added, there is an immediate decrease in the region of alpha-dispersion and an increase in the low frequency part of beta-dispersion. Similar changes are observed during ischemia following HTK perfusion without heptanol; additionally, the beta-dispersion shifts to higher frequencies. Using our models, we obtain analogue changes of epsilon (omega) by fitting model parameters which describe water content, water distribution, extra- and intracellular conductivity, and gap junction resistance. DISCUSSION: Changes of these tissue properties as calculated by our models based on the measurement of epsilon (omega) are consistent with intraischemic changes of heart tissue known from immunohistochemical, biochemical, and histological investigations. The next step will be to use our models for the prognosis of irreversible tissue damage.     

Highly Substituted Spiro[4.4]nonatrienes from a β-Amino-Substituted α,β-Unsaturated Fischer Carbene Complex and Three Molecules of an Arylalkyne

A novel mode of reaction towards arylethynes is shown by the β-trimethylsilyl-substituted α,β-unsaturated Fischer carbene complexes 1 . A mixture of the isomeric, highly substituted spiro[4.4]nonatrienes 2 and 3 is formed by the formal insertion of three alkyne molecules and subsequent cyclization (see scheme). Such selective triple insertions of alkynes into ethenylcarbene complexes have not been previously observed.     

Thermodynamic and electrochemical aspects ofp-tert-butylcalix[n]arenes (n=4, 6, 8) and their interactions with amines

Attention is drawn to the need of detailed thermodynamics in calixarene chemistry. The reasons for increasing efforts in this area are underlined and suggestions for new issues to be addressed are given. The solution thermodynamics ofp-tert-butylcalix[n]arenes (n=4, 6, 8) is discussed with particular reference to transfer Gibbs energies which reflect the selective solvation that the tetramer and the octamer undergo in the various solvents. This is followed by recent solution studies on amine-p-tert-butylcalix[n]arene (n=4, 6, 8) in nitrobenzene and in benzonitrile at 298.15 K which indicate the lower acidic character of the tetramer relative to the hexamer and the octamer in these solvents. As an implication of these results, very low conductivities are observed in studies involving the interaction of the former with amines. Thus, thermodynamic studies suggest thatp-tert-butylcalix[4]arene interacts with triethylamine in benzonitrile and in nitrobenzene through hydrogen bonding or ion-pair formation. A thermodynamic cycle is used to investigate the effect associated with the interaction of the amine with the tetramer in these solvents.This paper is dedicated to the commemorative issue on the 50th anniversary of calixarenes.     

Physiological aspects involved in production of xylanolytic enzymes by deep-sea hyperthermophilic archaeon Pyrodictium abyssi

Xylanases (EC3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. The hyperthermophilic archaeon Pyrodictium abyssi, which was originally isolated from marine hot abyssal sites, grows optimally at 97°C and is a prospective source of highly thermostable xylanase. Its endoxylanase was shown to be highly thermostable (over 100 m in at 105°C) and active even at 110°C. The growth of the deep-sea archaeon P. abyssi was investigated using different culture techniques. Among the carbohydrates used, beech wood xylan, birch wood glucuronoxylan and the arabinoxylan from oats pelt appeared to be good inducers for endoxylanase and β-xylosidase production. The highest production of arabinofuranosidase, however, was detected in the cell extracts after growth on xylose and pyruvate, indicating that the intermediate of the tricarboxylic acid cycle acted as a nonrepressing carbon source for the production of thi enzyme. Electron microscopic studies did not show a significant difference in the cell surface (e.g., xylanosomes) when P. abyssi cells were grown on different carbohydrates. The main kinetic parameters of the organism have been determined. The cell yield was shown to be very low owing to incomplete substrate utilization, but a very high maximal specific growth rate was determined (μ_max=0.0195) at 90°C and pH 6.0. We also give information on the problems that arise during the fermentation of this hyperthermophilic archaeon at elevated temperatures.     

Thermal and Photolytic Transformation of NHC–B,N‐Heterocycles: Controlled Generation of Blue Fluorescent 1,3‐Azaborinine Derivatives and 1H‐Imidazo[1,2‐a]indoles by External Stimuli

NHC–B,N‐heterocyclic compounds have been found to act as convenient precursors for obtaining either 1,3‐azaborinine or 1H‐imidazo[1,2‐a]indole derivatives, which are two different and rare classes of compounds. The formation of these two classes of compounds from the NHC–B,N‐heterocycles is highly selective depending on the external stimuli employed, and the resulting products have been studied for their interesting chemical and photophysical properties. The mechanism and possible reaction pathways of the unusual transformation are established by computational studies.     

Emerging Therapeutic Agents for Colorectal Cancer

There are promising new therapeutic agents for CRC patients, including novel small-molecule inhibitors and immune checkpoint blockers. We focused on emerging CRC’s therapeutic agents that have shown the potential for progress in clinical practice. This review provides an overview of tyrosine kinase inhibitors targeting VEGF and KIT, BRAF and MEK inhibitors, TLR9 agonist, STAT3 inhibitors, and immune checkpoint blockers (PD1/PDL-1 inhibitors), for which recent advances have been reported. These new agents have the potential to provide benefits to CRC patients with unmet medical needs.     

Tackling the chemical diversity of microbial nonulosonic acids – a universal large-scale survey approach

Nonulosonic acids, commonly referred to as sialic acids, are a highly important group of nine-carbon sugars common to all domains of life. They all share biosynthetic and structural features, but otherwise display a remarkable chemical diversity. In humans, sialic acids cover all cells which makes them important for processes such as cellular protection, immunity and brain development. On the other hand, sialic acids and other nonulosonic acids have been associated with pathological processes including cancer and viral infections. In prokaryotes, nonulosonic acids are commonly associated with pathogens, which developed through molecular mimicry a strategy to circumvent the host''s immune response. However, the remarkably large chemical diversity of prokaryotic nonulosonic acids challenges their discovery, and research on molecular characteristics essential for medical applications are often not feasible. Here, we demonstrate a novel, universal large-scale discovery approach that tackles the unmapped diversity of prokaryotic nonulosonic acids. Thereby, we utilize selective chemical labelling combined with a newly established mass spectrometric all-ion-reaction scanning approach to identify nonulosonic acids and other ulosonic acid-like sugars. In doing so, we provide a first molecular-level comparative study on the frequency and diversity across different phyla. We not only illustrate their surprisingly wide-spread occurrence in non-pathogenic species, but also provide evidence of potential higher carbon variants. Many biomedical studies rely on synthetic routes for sialic acids, which are highly demanding and often of low product yields. Our approach enables large-scale exploration for alternative sources of these highly important compounds.

A novel large-scale survey approach for microbial nonulosonic acids (sialic acids) including a first molecular level comparative study is presented.     

Chiral Sulfinamide Bisphosphine Catalysts: Design,Synthesis, and Application in Highly Enantioselective Intermolecular Cross‐Rauhut–Currier Reactions

A novel type of highly efficient chiral sulfinamide bisphosphine catalysts (Wei‐Phos) were developed. These could be easily prepared from commercially available starting materials. Wei‐Phos has shown good performance in the very challenging intermolecular cross‐Rauhut–Currier reactions of vinyl ketones and 3‐acyl acrylates or 2‐ene‐1,4‐diones, leading to the R‐C products in high yields with up to 99 % ee under 2.5–5 mol% catalyst loading. The highly regio‐ and enantio‐selective cross‐Rauhut–Currier reactions of 2‐ene‐1,4‐diones and vinyl ketone have yet reported so far.     

Chiral Sulfinamide Bisphosphine Catalysts: Design,Synthesis, and Application in Highly Enantioselective Intermolecular Cross‐Rauhut–Currier Reactions

A novel type of highly efficient chiral sulfinamide bisphosphine catalysts (Wei‐Phos) were developed. These could be easily prepared from commercially available starting materials. Wei‐Phos has shown good performance in the very challenging intermolecular cross‐Rauhut–Currier reactions of vinyl ketones and 3‐acyl acrylates or 2‐ene‐1,4‐diones, leading to the R‐C products in high yields with up to 99 % ee under 2.5–5 mol% catalyst loading. The highly regio‐ and enantio‐selective cross‐Rauhut–Currier reactions of 2‐ene‐1,4‐diones and vinyl ketone have yet reported so far.     

A new scale of electronegativity based on electrophilicity index

By calculating the energies of neutral and different ionic forms (M2+, M+, M, M-, and M2-) of 32 elements (using B3LYP/6-311++G** level of theory) and taking energy (E) to be a Morse-like function of the number of electrons (N), the electrophilicity values (omega) are calculated for these atoms. The obtained electrophilicities show a good linearity with some commonly used electronegativity scales such as Pauling and Allred-Rochow. Using these electrophilicities, the ionicities of some diatomic molecules are calculated, which are in good agreement with the experimental data. Therefore, these electrophilicities are introduced as a new scale for atomic electronegativity, chi(omega)0. The same procedure is also performed for some simple polyatomic molecules. It is shown that the new scale successfully obeys Sanderson's electronegativity equalization principle and for those molecules which have the same number of atoms, the ratio of the change in electronegativity during the formation of a molecule from its elements to the molecular electronegativity (Delta chi/chi omega) is the same.     

Antiinfluenza virus activity of a bacteriocin produced by Lactobacillus delbrueckii

A novel antibacterial substance produced by Lactobacillus delbrueckii has been isolated and characterized (1). The inhibitory agent corresponded to the criteria for bacteriocins. It was active against lactic acid bacteria (LAB) species and several food-borne pathogens. The cell-free supernatant was purified by HPLC gel-filtration. Three preparations at different purification steps were tested for activity on the reproduction of influenza virus A/chicken/Germany, strain Weybridge (H7N7) and strain Rostock (H7N1) in cell cultures of chicken embryo fibroblasts (CEF). The inhibitory effect was shown to be highly selective and specific. Expression of viral glycoproteins hemagglutinin, neuraminidase, and nucleoprotein on the surface of infected cells, virus-induced cytopathic effect, infectious virus yield, and hemagglutinin production were all reduced at nontoxic concentrations of the crude preparation (B1). B1 did not protect cells from infection, did not affect adsorption, and slightly inhibited viral penetration into infected cells. The purification did not enhance the cellular toxicity and increased about 870-fold the virus-inhibitory activity. No inactivating effect on extracellular virus was found.