Stage-specific embryonic antigen 3 as a marker of visceral extraembryonic endoderm

The distribution of the stage-specific embryonic antigen SSEA-3 was studied immunohistochemically on postimplantation mouse embryos. This carbohydrate antigen, identified as an epitope of a globo-series ganglioside isolated from human teratocarcinoma cells (Kannagi et al., 1983, J. Biol. Chem.258, 8934–8942) was originally detected on the zygote and mouse early cleavage-stage embryos. It disappears on the early blastocyst and reappears on the primitive endoderm of the implanting blastocyst (Shevinsky et al., 1982, Cell30, 697–705). We now show in the early egg cylinder (on the sixth day of pregnancy) SSEA-3 is present in the entire visceral endoderm but not in any other part of the conceptus. From Day 7 of pregnancy onward, SSEA-3 is restricted to the extraembryonic visceral endoderm and the visceral yolk sac cells. Therefore, SSEA-3 is a useful marker for this endodermal cell lineage in midgestational mouse embryos.     

Flicker-response enhancement in ‘fast-eyed’ insects

When subjected to a sequence of light flashes of the same duration and intensity Drosophila melanogaster and Calliphora erythrocephala have been found to display a frequency-dependent alternating pattern of ERG response with one subset of flicker-responses being amplified (enhanced) and the other subset being reduced in amplitude-often to zero (Hamdorfet al., 1969, Z. vergl. Physiol.63, 91–112; Cosens and LeBlanc, 1980, J. Comp. Physiol.137, 341–351). It has been suggested (Fouchard and Carricaburu, 1972, J. Comp. Physiol.77, 341–355) that this response pattern is an artefact due to the successive flashes not actually being equivalent.In this study we show that the alternation of flicker-responses seen in ‘slow-eyed’ insects by Fouchard and Carricaburu is different in nature from that seen in the ‘fast-eyed’ insects. In the latter case the phenomenon is shown to be physiological (as Hamdorfet al., claimed) with a dependence on stimulus intensity and contrast with background illumination. However, even differences in successive flashes facilitate response enhancement and also the appearance of a secondary alternation of the amplified subset of responses. Through response enhancement both the critical flicker fusion frequency and the response amplitude at an enhancing frequency, are increased.     

A quantitative description of membrane current and its application to conduction and excitation in nerve

This article concludes a series of papers concerned with the flow of electric current through the surface membrane of a giant nerve fibre (Hodgkinet al., 1952,J. Physiol. 116, 424–448; Hodgkin and Huxley, 1952,J. Physiol. 116, 449–566). Its general object is to discuss the results of the preceding papers (Section 1), to put them into mathematical form (Section 2) and to show that they will account for conduction and excitation in quantitative terms (Sections 3–6).     

Hydrophilic and hydrophobic attachment of both globular and asymmetric acetylcholinesterase to frog muscle basal lamina sheaths

We prepared myofiber basal lamina sheaths (BLs) using the in vivo experimental procedure of Sanes et al. (J. Cell Biol.78, 176–198, 1978) on frog cutaneus pectoris muscle. On the 15 days post-operatively, acetylcholinesterase (AChE) is still found concentrated in native BLs and purified BLs preparations and both globular and asymmetric molecular forms coexist (Nicolet et al., J. Cell Biol., 107, 762–768, 1986). We describe here at least two distinct AChE pools, according to their differential solubility in non-ionic detergent and high-salt media. One is detergent-extracted (DE) and the other is detergent-insoluble, high-salt extracted (HSS). In the BLs preparation as well as in control motor end-plate rich regions (MEP-r) of muscle, both globular and asymmetric forms of AChE are found as DE and HSS variants. These observations suggest that all AChE forms are present in the extracellular muscle basal lamina and are bound through not only hydrophilic but also hydrophobic bonds, to probably distinct structural domains of the muscle basal lamina.     

Synthesis and biological evaluation of imidazole derivatives as novel NOP/ORL1 receptor antagonists: Exploration and optimization of alternative pyrazole structure

Nonpeptidic small-molecule NOP/ORL1 receptor antagonists with an imidazole scaffold were designed and synthesized to investigate alternatives to the pyrazole analog. Systematic modification of the original pyrazole lead [Kobayashi et al., Bioorg. Med. Chem. Lett. 2009, 19, 3627; Kobayashi et al., Bioorg. Med. Chem. Lett., in press] to change the heterocyclic core, substituted side chain, and pendant functional group demonstrated that examining the structure–activity relationship for novel templates allowed the identification of potent, fully substituted 4-aminomethyl-1H-imidazole and 2-aminomethyl-1H-imidazole. These compounds exhibited excellent potency for ORL1 receptor with minimal P-gp efflux and/or reduced hERG affinity.     

Three-Dimensional Reconstruction of the Mammalian Centriole from Cryoelectron Micrographs: The Use of Common Lines for Orientation and Alignment

The microtubule organizing center of the animal cell (S. D. Fulleret al.,1992,Curr. Opin. Struct. Biol.2,264–274; D. M. Gloveret al.,1993,Sci. Am.268,62–68; E. B. Wilson, 1925), (The Cell in Development and Heredity) comprises two centrioles and the pericentriolar material. We have completed several three-dimensional reconstructions of individual centrioles from tilt series of cryoelectron micrographs. The reconstruction procedure uses minimization of the common lines residual to define the orientation of the centriolar ninefold symmetry axis and then uses this symmetry to generate a structure by weighted backprojection to 28-nm resolution. Many of the features of these reconstructions agree with previous, conventional transmission electron microscopy studies (M. Paintrandet al.,1992,J. Struct. Biol.108,107–128). The microtubule barrel of the centriole is roughly 500 nm long and 300 nm in diameter and the microtubule bundles appear to taper toward the distal end. In addition, we see a handedness to the pericentriolar material at the base (distal end) of the centriole which is opposite to the skew of the microtubule triplets. The region at which the microtubule barrel joins this base is intriguingly complex and includes an internal cylindrical feature which is a site of γ tubulin localization.     

Sterically induced conformational restriction: Discovery and preclinical evaluation of novel pyrrolo[3,2-d]pyrimidines as microtubule targeting agents

The discovery, synthesis and biological evaluations of a series of nine N5-substituted-pyrrolo[3,2-d]pyrimidin-4-amines are reported. Novel compounds with microtubule depolymerizing activity were identified. Some of these compounds also circumvent clinically relevant drug resistance mechanisms (expression of P-glycoprotein and βIII tubulin). Compounds 4, 5, and 8–13 were one to two-digit nanomolar (IC₅₀) inhibitors of cancer cells in culture. Contrary to recent reports (Banerjee et al. J. Med. Chem.2018, 61, 1704–1718), the conformation of the most active compounds determined by ¹H NMR and molecular modeling are similar to that reported previously and in keeping with recently reported X-ray crystal structures. Compound 11, freely water soluble as the HCl salt, afforded statistically significant inhibition of tumor growth in three xenograft models [MDA-MB-435, MDA-MB-231 and NCI/ADR-RES] compared with controls. Compound 11 did not display indications of animal toxicity and is currently slated for further preclinical development.     

Discovery of 3-morpholino-imidazole[1,5-a]pyrazine BTK inhibitors for rheumatoid arthritis

8-Amino-imidazo[1,5-a]pyrazine-based Bruton’s tyrosine kinase (BTK) inhibitors, such as 6, exhibited potent inhibition of BTK but required improvements in both kinase and hERG selectivity (Liu et al., 2016; Gao et al., 2017). In an effort to maintain the inhibitory activity of these analogs and improve their selectivity profiles, we carried out SAR exploration of groups at the 3-position of pyrazine compound 6. This effort led to the discovery of the morpholine group as an optimized pharmacophore. Compounds 13, 23 and 38 displayed excellent BTK potencies, kinase and hERG selectivities, and pharmacokinetic profiles.     

Chemical constituents of Peperomia tetraphylla (Forst. F.) Hooker et Arnott

Sixteen compounds were isolated from the whole herbs of Peperomia tetraphylla (Forst. F.) Hooker et Arnott by phytochemical methods, including eight flavonoids (1–3, 6, 7, 14–16), three lignans (8–10), three beta sitosterols (4, 5, 11), and two phenolic acids (12, 13). Their structures were identified by the analysis of NMR and MS, as well as the comparisons to the reported data. Among them, 2″-O-xylosylisoswertisin (14) was firstly isolated from the Piperaceae family, as well as ten compounds (1–4, 7, 10–11, 13, 15–16) were isolated from P. tytraphylla for the first time. Moreover, the chemotaxonomic significance of constituents isolated from P. tytraphylla was also discussed.     

Regulation of glutamine synthetase adenylylation and deadenylylation by the enzymatic uridylylation and deuridylylation of the PII regulatory protein

Regulation of glutamine synthetase activity in Escherichia coli is mediated by covalent attachment and detachment of an adenylyl group to each subunit of the enzyme [Kingdon, H. S. et al., Proc. Nat. Acad. Sci., 58, 1703, (1967); Wulff, K. D. et al., Biochem. Biophys. Res. Commun.28, 740, (1967)]. Adenylylation and deadenylylation of the enzyme are both catalyzed by a single adenylyltransferase (ATase) whose activity is modulated by various metabolites and by a regulatory protein, P_II [Shapiro, B. M., Biochemistry; Anderson, W. B. et al., Proc. Nat. Acad. Sci.67, 1761 (1970)].The present study confirms preliminary results [Brown, M. S. et al., Proc. Nat. Acad. Sci.68, 2949 (1971)] showing that: (1) the regulatory protein (P_II) exists in two interconvertible forms, P_IIA and P_IID, which, respectively, stimulate adenylylation and deadenylylation activity of ATase; (2) conversion of P_IIA to P_IID requires the presence of UTP, 2-oxoglutarate, ATP, and either Mg²⁺ or Mn²⁺; (3) this conversion involves covalent attachment of a uridine derivative to P_IIA. It is further established that the covalently bound uridine derivative is UMP which is derived from UTP in a reaction catalyzed by a specific uridylyltransferase (UTase). Removal of the covalently bound UMP from P_IID is catalyzed by a separate enzyme, referred to as the uridylyl-removing enzyme (UR-enzyme). This enzyme has an obligatory requirement for Mn²⁺.Regulation of glutamine synthetase activity in E. coli is thus facilitated by a highly sophisticated cascade system of proteins, consisting of an ATase, the regulatory protein (P_II), UTase, and the UR-enzyme. The activities of these various components is rigorously controlled by various metabolites, including glutamine, 2-oxoglutarate, ATP, P_i, UTP, and the divalent cations, Mn²⁺ and Mg²⁺.     

Thymosin β4 in cultured mammalian cell lines

Thymosin β₄, originally isolated from calf thymus [Low et al., Proc. Nat. Acad. Sci. USA78, 1162–1166 (1981)] is present in a number of cell lines unrelated to the reticuloendothelium, including myoblasts and fibroblasts. It is also actively synthesized by these cell lines. Its content and rate of synthesis in the cell lines examined appear to be correlated with their ability to adhere and their motility.     

Structural investigations of recombinant urokinase growth factor-like domain

Urokinase-type plasminogen activator (uPA) is a serine protease that converts the plasminogen zymogen into the enzymatically active plasmin. uPA is synthesized and secreted as the single-chain molecule (scuPA) composed of an N-terminal domain (GFD) and kringle (KD) and C-terminal proteolytic (PD) domains. Earlier, the structure of ATF (which consists of GFD and KD) was solved by NMR (A. P. Hansen et al. (1994) Biochemistry, 33, 4847–4864) and by X-ray crystallography alone and in a complex with the soluble form of the urokinase receptor (uPAR, CD87) lacking GPI (C. Barinka et al. (2006) J. Mol. Biol., 363, 482–495). According to these data, GFD contains two β-sheet regions oriented perpendicularly to each other. The area in the GFD responsible for binding to uPAR is localized in the flexible Ω-loop, which consists of seven amino acid residues connecting two strings of antiparallel β-sheet. It was shown by site-directed mutagenesis that shortening of the Ω-loop length by one amino acid residue leads to the inability of GFD to bind to uPAR (V. Magdolen et al. (1996) Eur. J. Biochem., 237, 743–751). Here we show that, in contrast to the above-mentioned studies, we found no sign of the β-sheet regions in GFD in our uPA preparations either free or in a complex with uPAR. The GFD seems to be a rather flexible and unstructured domain, demonstrating in spite of its apparent flexibility highly specific interaction with uPAR both in vitro and in cell culture experiments. Circular dichroism, tryptophan fluorescence during thermal denaturation of the protein, and heteronuclear NMR spectroscopy of ¹⁵N/¹³C-labeled ATF both free and in complex with urokinase receptor were used to judge the secondary structure of GFD of uPA.     

The measurement of partial specific volumes and sedimentation coefficients by sucrose density centrifugation

The method of Martin and Ames (1961, J. Biol. Chem.236, 1372) gives good estimates of the s_20,w of proteins when the SW40 rotor is used with a sucrose gradient. Viscosities of sucrose in D₂O were measured, and the data were used in computer simulations to test alternate approaches to estimating $\text{v}$ and s_20,w values by comparisons with standards. The method of Meunier et al. (1972, FEBS Lett.24, 63) for $\text{v}$ was shown to be optimal. For s_20,w estimations, substantial errors were found with the methods of Bon et al. (1973, Eur. J. Blochem.35, 372) and especially Meunier et al. When standards and unknowns have the same $\text{v}$, and the gradient is made up in water or dilute buffer, the simple ratio method of Martin and Ames gives most accurate results for s_20,w. For all other cases, an alternative procedure is described.     

Refinement of an adherence assay for the measurement of bacterial attachment to silica beads

An in vitro adherence assay [4] which utilised radiolabelled bacteria and silica beads was used to investigate firm and loose attachment of bacteria. A systematic evaluation of the effects of the assay conditions, including incubation vessel, bacteria to bead interaction, bead washing and bead weight, was carried out in an attempt to improve the reproducibility of adherence measurements. Four oral streptococcal strains (S. mutans NCTC 10449, S. sanguis NCTC 7863 and 10904, S. salivarius NCTC 8618) were studied with saliva coated and uncoated silica beads.Using the improved system, total bacterial adherence to uncoated beads was high (45–60% of bacteria present). While saliva coating of beads reduced total and firm adherence of all strains, these reductions were less marked (P > 0.005) for S. sanguis NCTC 10904.     

Analysis of partial assortative mating and sexual selection models for a polygamous species involving a trait based on levels of heterozygosity

The consequences of preferential mating in the presence of partial assortative and sexual selection mechanisms are ascertained for a two-allele one-locus trait involving two phenotype classes C₁ = {all homozygotes} and C₂ = {heterozygotes}. Relevant biological cases may include Burley (1977, Proc. Nat. Acad. Sci. USA74, 3476–3479), Wilbur et al. (1978, Evolution32, 264–270), and Singh and Zouros (1978, Evolution32, 342–353). When the preference rate for the heterozygote exceeds that for homozygotes, it is established that the unique stable state is the central Hardy-Weinberg equilibrium. The rate of approach is faster with sexual selection than for the corresponding model of assortative mating. When the preference rates favor the homozygotes then in this symmetric model of sexual selection two asymmetric Hardy-Weinberg polymorphisms can evolve, and which succeeds depends on initial conditions. The models are also analyzed with natural selection acting on phenotypes superimposed on assortative mating. In this case we can have up to three coexisting stable states involving both fixation alternatives and a central polymorphism. The corresponding model with sexual selection maintains either the central equilibrium as in assortative mating or two asymmetric polymorphic equilibria.     

Preparation of Biologically Active Recombinant Human Progastrin1–80

The bacterial expression of human progastrin_6–80 has been reported previously [Baldwin, G.S. et al. (2001) J. Biol. Chem. 276: 7791-7796]. The aims of the present study were to prepare full-length recombinant human progastrin_1–80 and to compare its biological activity with that of progastrin_6–80 in vitro, to determine whether or not the N-terminal five amino acids contributed to activity. A fusion protein of glutathione-S-transferase and human progastrin_1–80 was expressed in Escherichia coli, collected on glutathione-agarose beads, and cleaved with enterokinase. Progastrin_1–80 was purified by reversed-phase and anion exchange HPLC and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. No differences were detected in the extent of stimulation by progastrin_1–80 and progastrin_6–80 in proliferation and migration assays with the mouse gastric cell line IMGE-5. We conclude that residues 1–5 of progastrin_1–80 are not essential for biological activity.     

Determination of membrane-bound chloroplast RNA by the ethidium bromide fluorometric method

The method of El-Hamalawi et al. [(1975) Anal. Biochem.67, 384–391] for the fluorometric determination of nucleic acids with ethidium bromide has been adapted for the assay of membrane-associated chloroplast RNA. Membranes are stripped of RNA by incubation in a high-salt buffer lacking Mg²⁺, and the RNA is collected by magnesium phosphate-ethanol coprecipitation. RNA levels are determined by measuring the degree of enhancement of ethidium bromide fluorescence.     

Design,synthesis, and opioid activity of arodyn analogs cyclized by ring-closing metathesis involving Tyr(allyl)

Kappa (κ) opioid receptor selective antagonists are useful pharmacological tools in studying κ opioid receptors and have potential to be used as therapeutic agents for the treatment of a variety of diseases including mood disorders and drug addiction. Arodyn (Ac[Phe^1–3,Arg⁴,d-Ala⁸]Dyn A-(1–11)NH₂) is a linear acetylated dynorphin A (Dyn A) analog that is a potent and selective κ opioid receptor antagonist (Bennett et al. J Med Chem 2002;45:5617–5619) and prevents stress-induced reinstatement of cocaine-seeking behavior following central administration (Carey et al. Eur J Pharmacol 2007;569:84–89). To restrict its conformational mobility, explore possible bioactive conformations and potentially increase its metabolic stability we synthesized cyclic arodyn analogs on solid phase utilizing a novel ring-closing metathesis (RCM) reaction involving allyl-protected Tyr (Tyr(All)) residues. This approach preserves the aromatic functionality and directly constrains the side chains of one or more of the Phe residues. The novel cyclic arodyn analog 4 cyclized between Tyr(All) residues incorporated in positions 2 and 3 exhibited potent κ opioid receptor antagonism in the [³⁵S]GTPγS assay (K_B?=?3.2?nM) similar to arodyn. Analog 3 cyclized between Tyr(All) residues in positions 1 and 2 also exhibited nanomolar κ opioid receptor antagonist potency (K_B?=?27.5?nM) in this assay. These are the first opioid peptides cyclized via RCM involving aromatic residues, and given their promising pharmacological activity represent novel lead peptides for further exploration.     

Synthesis and in vitro reactivation study of isonicotinamide derivatives of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide as reactivators of Sarin and VX inhibited human acetylcholinesterase (hAChE)

Previously (Karade et al., 2014), we have reported the synthesis and in vitro evaluation of bis-pyridinium derivatives of pyridine-3-yl-(2-hydroxyimino acetamide), as reactivators of sarin and VX inhibited hAChE. Few of the molecules showed superior in vivo protection efficacy (mice model) (Kumar et al., 2014; Swami et al., 2016) in comparison to 2-PAM against DFP and sarin poisoning. Encouraged by these results, herein we report the synthesis and in vitro evaluation of isonicotinamide derivatives of pyridine-3-yl-(2-hydroxyimino acetamide) (4a–4d) against sarin and VX inhibited erythrocyte ghost hAChE. Reactivation kinetics of these compounds was studied and the determined kinetic parameters were compared with that of commercial reactivators viz. 2-PAM and obidoxime. In comparison to 2-PAM and obidoxime, oxime 4a and 4b exhibited enhanced reactivation efficacy toward sarin inhibited hAChE while oxime 4c showed far greater reactivation efficacy toward VX inhibited hAChE. The acid dissociation constant and IC₅₀ values of these oximes were determined and correlated with the observed reactivation potential.     

Three New Species of Predatory Nematodes (Dorylaimida: Actinolaimoidea) from Korea

Three new species of predatory nematodes belonging to family Actinolaimidae, superfamily Actinolaimoidea, order Dorylaimida are described and illustrated. Westindicus sanaensis sp. n. has a body 1.9–2.4mm long; b = 3.7–4.3; c = 8.1–8.5; odontostyle 26.5–28.0μm long; spicules 71–74μm long; ventromedian supplements 15 and comes close to Westindicus cheongsongensis Choi et al, 1998 and Westindicus keralaensis Khan et al., 1994. Egtitus koriensis sp. n. is 1.8–2.0 mm long; b = 3.4–4.1; c = 14–18; odontostyle 20–21 μm, and comes close to Egtitus elaboratus (Cobb, 1906) Thorne, 1967; Egtitus itanagrus Khan et al., 1994 and Egtitus adhricus Khan & Jairajpuri, 1994. Stopractinca glandulus sp. n. is 2.3–2.4 mm long; b = 4.0–4.1; c = 11–12; odontostyle 26–28μm and differs from other two known species viz., Stopractincta orientalis Khan et al., 1994 and Stopractincta malnadensis Dhanam et al., 1994 in having very thick body cuticle and longer cardia, and in the presence of a gland at the base of oesophagus.