Synthesis,antimalarial and antitubercular activity of acetylenic chalcones

A series of acetylenic chalcones were evaluated for antimalarial and antitubercular activity. The antimalarial data for this series suggests that growth inhibition of the W2 strain of Plasmodium falciparum can be imparted by the introduction of a methoxy group ortho to the acetylenic group. Most compounds were more active against non-replicating than replicating cultures of Mycobacterium tuberculosis H₃₇Rv, an unusual pattern with respect to existing anti-TB agents.     

Quantitative effects of unsaturated fatty acids in microbial mutants. VII. Influence of the acetylenic bond location on the effectiveness of acyl chains.

The ability of a series of 18 carbon acetylenic fatty acids to fulfill the unsaturated fatty acid requirements of Escherichia coli and Saccharomyces cerevisiae was investigated. Despite their high melting points (greater than 40 degrees C), several isomers of the acetylenic fatty acids were as efficient or more efficient in supporting growth than the analogous fatty acid having a cis-double bond. The efficiencies of the different positional isomers in supporting cell proliferation varied from essentially 0 cells per fmol for the 2-5 and 13-17 isomers to high values when the acetylenic bond was near the center of the chain: e.g. 45 E. coli and 5.5 S. cerevisiae cells/fmol for the 10 isomer. A striking ineffectiveness of the 9 isomer was observed with E. coli. The 7, 8 and 10 isomers were at least 10-fold more efficient than any of the other positional isomers in supporting the growth of E. coli. In contrast, the 9 isomer was among the most effective acetylenic fatty acids tested with the yeast mutant. Chromatographic analysis of the extracted lipids indicated that each of the acetylenic isomers tested (except delta2 and delta3) could be esterified by the prokaryotic and eukaryotic microorganisms. The content of unsaturated plus cyclopropane acids observed when growth ceased in E. coli cultures supplemented with growth-limiting concentrations of the acetylenic fatty acids ranged from approx. 15 mol% for the 8 isomer to approx. 35 mol% for the 14 and 17 isomers. The 8-11 isomers were observed to be esterified predominantly at the two position in phosphatidylethanolamine of E. coli and in phosphatidylcholine of S. cerevisiae.     

Long-chain triazolyl acids as inhibitors of osteoclastogenesis

Saturated fatty acids (e.g., palmitic acid) are known to moderately inhibit the development of osteoclasts in vitro. In pursuit of more effective inhibitors of osteoclastogenesis we explored two new classes of palmitic acid analogues containing either an ether or triazolyl group at various positions along the chain. The compounds were evaluated for their ability to inhibit the formation of osteoclasts in primary mouse bone marrow cultures. The oxyacids were generally prepared by condensation of the appropriate alkyl halides and diols, followed by Jones oxidation. The triazolyl acids were prepared by copper-catalysed click chemistry between alkyl azides and acetylenic acids, or with the appropriately-protected azides and alkynes, followed by deprotection and oxidation. The oxyacids were little more effective than palmitic acid, but the triazolyl analogues were much more effective osteoclastogenesis inhibitors, especially when the triazole was distant from the acid unit.     

Lipids in plant tissue cultures. II. Unusual fatty acids in lipids of Hydnocarpus anthelminthica cultures

The lipids in callus cultures of Hydnocarpus anthelminthica were studied after 60, 160 and 460 days of growth. In each of the cultures the lipid classes usually found in plant tissue cultures were detected. With increasing age of the cultures the total lipid content as well as the proportions of triglycerides decreased. The major constituent fatty acids of the total lipids were palmitic and linoleic acids. Small amounts of cyclopentenyl fatty acids were also present. The proportions of saturated straight-chain fatty acids increased with the age of the cultures whereas the proportions of monounsaturated straight-chain fatty acids decreased. Only small changes were observed with polyunsaturated fatty acids. The content of cyclopentenyl fatty acids rose with the age of the cultures. The monounsaturated straight-chain fatty acids consisted of mixtures of isomers whose composition changed with the age of the cultures. In contrast, the polyunsaturated straight-chain fatty acids belonged exclusively to the Δ9 series, regardless of the age of the cultures.     

ADAM10 mediates N‐cadherin ectodomain shedding during retinal ganglion cell differentiation in primary cultured retinal cells from the developing chick retina

Here, we examined the role of ADAM10 during retinal cell differentiation in retinal sections and in vitro cultures of developing chick retinal cells from embryonic day 6 (ED6). Immunohistochemistry showed that ADAM10 is abundantly expressed in the inner zone of neuroblastic layer at ED5, and it becomes more highly expressed in the ganglion cell layer at ED7 and ED9. Western blotting confirmed that ADAM10 was expressed as an inactive pro‐form that was processed to a shorter, active form in control cultured cells, but in cultures treated with an ADAM10 inhibitor (GI254023X) and ADAM10‐specific siRNA, the level of mature ADAM10 decreased. Phase‐contrast microscopy showed that long neurite extensions were present in untreated cultures 24 h after plating, whereas cultures treated with GI254023X showed significant decreases in neurite extension. Immunofluorescence staining revealed that there were far fewer differentiated ganglion cells in ADAM10 siRNA and GI254023X‐treated cultures compared to controls, whereas the photoreceptor cells were unaltered. The Pax6 protein was more strongly detected in the differentiated ganglion cells of control cultures compared to ADAM10 siRNA and GI254023X‐treated cultures. N‐cadherin ectodomain shedding was apparent in control cultures after 24 h, when ganglion cell differentiation was observed, but ADAM10 siRNA and GI254023X treatment inhibited these processes. In contrast, N‐cadherin staining was strongly detected in photoreceptor cells regardless of ADAM10 siRNA and GI254023X treatment. Taken together, these data indicate that the inhibition of ADAM10 can inhibit Pax6 expression and N‐cadherin ectodomain shedding in retinal cells, possibly affecting neurite outgrowth and ganglion cell differentiation. J. Cell. Biochem. 114: 942–954, 2013. © 2013 Wiley Periodicals, Inc.     

Facile synthesis of substituted thiophenes via Pd/C-mediated sonogashira coupling in water

The first successful Pd/C-mediated Sonogashira coupling of iodothiophene with terminal alkynes in water is described here. Pd/C-CuI-PPh₃ was found to be an efficient catalyst system for this coupling reaction. Using this economic and reliable process a variety of acetylenic thiophenes with a wide range of functional groups were prepared in good yields. Synthetic applications and in vitro anticancer properties of some of the compounds synthesized are described.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum.

A study was made of the time course and kinetics of [3H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [3H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent Kmvalues for GABA neurons for high and low affinity uptake were 0.33 X 10(-6) M and 41.8 X 10(-4) M, respectively. For nonneuronal cells, the apparent Km for high affinity uptake was 0.29 X 10(-6) M. The apparent Vmax values for GABA neurons for high and low affinity uptake were 28.7 X 10(-6) mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent Vmax for high affinity uptake was 0.06 X 10(-6) mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 X 10(-9) M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [3H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by noneuronal cells was only slightly decreased. Most (75-85%) of the [3H]GABA (4.4 X 10(-6) M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

In situ imaging reveals properties of purinergic signalling in trigeminal sensory ganglia in vitro

Chronic pain is supported by sterile inflammation that induces sensitisation of sensory neurons to ambient stimuli including extracellular ATP acting on purinergic P2X receptors. The development of in vitro methods for drug screening would be useful to investigate cell crosstalk and plasticity mechanisms occurring during neuronal sensitisation and sterile neuroinflammation. Thus, we studied, at single-cell level, membrane pore dilation based on the uptake of a fluorescent probe following sustained ATP-gated P2X receptor function in neurons and non-neuronal cells of trigeminal ganglion cultures from wild-type (WT) and R192Q Ca_V2.1 knock-in (KI) mice, a model of familial hemiplegic migraine type 1 characterised by neuronal sensitisation and higher release of soluble mediators. In WT cultures, pore responses were mainly evoked by ATP rather than benzoyl-ATP (BzATP) and partly inhibited by the P2X antagonist TNP-ATP. P2X7 receptors were expressed in trigeminal ganglia mainly by non-neuronal cells. In contrast, KI cultures showed higher expression of P2X7 receptors, stronger responses to BzATP, an effect largely prevented by prior administration of Ca_V2.1 blocker ω-agatoxin IVA, small interfering RNA (siRNA)-based silencing of P2X7 receptors or the P2X7 antagonist A-804598. No cell toxicity was detected with the protocols. Calcitonin gene-related peptide (CGRP), a well-known migraine mediator, potentiated BzATP-evoked membrane permeability in WT as well as R192Q KI cultures, demonstrating its modulatory role on trigeminal sensory ganglia. Our results show an advantageous experimental approach to dissect pharmacological properties potentially relevant to chronic pain and suggest that CGRP is a soluble mediator influencing purinergic P2X pore dilation and regulating inflammatory responses.     

Explanted Fish Neural Tubes Give Rise to Differentiating Neural Crest Cells

Neural tubes were explanted from the trunk of various embryonic stages of three teleost fish, Xiphophorus maculatus (platyfish), X. helleri (swordtail), and Oryzias latipes (Japanese medaka) with the aim to obtain in vitro differentiating neural crest cells. Outgrowth of cells was observed immediately after attachment of the explants on dishes coated with fibronectin. The outgrowing cells stained with the HNK-1 monoclonal antibody indicating that they were neural crest cells. Maximum cell outgrowth was obtained from explants of stage 9 of Xiphophorus and 19 of medaka, i.e., from stages characteristic of maximal neural crest cell segregation, and by the use of Leibovitz's (L-15) medium supplemented with 20% FBS. In this medium cells survived for more than two weeks; M199 also gave satisfactory results but DMEM allowed only poor cell growth and survival. Neuronal cells could be observed in all cultures after 48 hr, in some medaka cultures these cells were mixed with pigment cells but homogeneous pigment cell cultures were also observed. This in vitro system will be invaluable for the study of the developmental potential of fish neural crest cells and the contributions of extrinsic factors in neural crest cell fate.     

Alkamides: a critical reconsideration of a multifunctional class of unsaturated fatty acid amides

Alkamides are natural products formed by connecting straight-chain, mostly unsaturated, aliphatic acids with various amines by an amide linkage. More than 300 derivatives are known from eight plant families consisting of various combinations of 200 acids with 23 amines. Apart from a few saturated derivatives alkamides with unsaturated acid parts are grouped into compounds with purely olefinic patterns and those with olefinic and acetylenic linkages. Derived from C₁₈ oleic acid the acid parts are modified either by chain elongations to C₂₈ or by oxidative shortenings to C₄ acid residues. Substrate and regiospecific desaturases and acetylenases are responsible for their characteristic patterns of unsaturation. Amine parts are derived from various amino acids by decarboxylation. Beside the widespread isobutylamines alkamides with six- and five-membered ring amines and those with phenylalanine derived amines are characteristic for the Asteraceae and Piperaceae while benzylamines are restricted to the Brassicaceae. Within the Asteraceae 2-methylbutylamine distinguishes the tribe Heliantheae from Anthemideae characterized by ring amines. Alkamides with elongated olefinic acid parts are mainly found in Piperaceae and Brassicaceae while acetylenic acid parts are typical for Asteraceae. A wide variety of biological activities ranges from the characteristic pungent/tingling property and high insecticidal toxicity to significant antifungal, antibacterial, antiprotozoal, molluscicidal, cercaricidal, and acaricidal activity. They also act as plant growth-promoting substances. Position and stereochemistry of the double bonds are essential for the different qualities of the pungent taste. Medically alkamides possess anti-inflammatory and analgesic properties and are responsible for immuno-modulatory and cannabinomimetic effects.     

Studies of the temporal relationship between the cytogenetic and biochemical manifestations of X-chromosome inactivation during the differentiation of LT-1 teratocarcinoma stem cells

Previous biochemical studies have suggested that both X chromosomes produce gene products when cells of the LT-1 teratocarcinoma stem cell line are maintained in the undifferentiated state, and that dosage compensation, the biochemical manifestation of X inactivation, occurs when the cells are induced to differentiate in vitro (Martin et al., 1978). In this study the differentiation of LT-1 cells in vitro is described in detail, and data from cytogenetic studies of the time of X-chromosome replication in LT-1 cells are presented. They show that as long as the cells are maintained in the undifferentiated state both X chromosomes in each cell show the isocyclic replication pattern typical of a genetically active chromosome. However, when the LT-1 cells are induced to differentiate under appropriate conditions, one of the two X chromosomes in each cell of a large proportion of the population displays the allocyclic (either early or late) replication pattern typical of an inactive X chromosome. These data thus confirm that undifferentiated LT-1 cells contain two active X chromosomes and that X inactivation occurs in differentiating cultures of LT-1 cells. It is further demonstrated that there is a close temporal correlation between the biochemical and cytogenetic manifestations of the X-inactivation process. In addition, we observed that although X inactivation does not occur in the absence of morphological differentiation, it does not always occur when the cells differentiate in vitro.     

Infection of human T lymphocytes with varicella-zoster virus: an analysis with viral mutants and clinical isolates

Varicella-zoster virus (VZV) disseminates in the body in peripheral blood mononuclear cells during chickenpox. Up to 1 in 10,000 mononuclear cells are infected during the viremic phase of the disease. We developed an in vitro system to infect human mononuclear cells with VZV by using umbilical cord blood. In this system, 3 to 4% of T cells were infected with VZV. VZV mutants unable to express certain genes, such as open reading frame 47 (ORF47) or ORF66, were impaired for growth in T cells, while other mutants showed little difference from parental virus. VZV unable to express ORF47 was even more impaired for spread from umbilical cord blood cells to melanoma cells in vitro. Early-passage clinical isolates of VZV infected T cells at a similar rate to the Oka vaccine strain; however, the clinical isolates were more efficient in spreading from infected T cells to melanoma cells. This in vitro system for infecting human T cells with VZV should be useful for identifying cellular and viral proteins that are important for virus replication in T cells and for the spread of virus from T cells to other cells.     

Antitumor compound testing in glioblastoma organotypic brain cultures

Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumor. Identification of new therapeutic regimens is urgently needed. A major challenge remains the development of a relevant in vitro model system with the necessary capacity and flexibility to profile compounds. The authors have developed and characterized a 3D culture system of brain cells (brain Hi-Spot) where GBM-derived cells can be incorporated (GBM/brain Hi-Spot). Immuno-fluorescence and electrophysiological recordings demonstrate that brain Hi-Spots recapitulate many features of brain tissue. Within this tissue, GBM-derived cell growth is monitored using a fluorescence assay. GBM-derived cells growing in Hi-Spots form tumor nodules that display properties of GBM such as 5-Ala positive staining, an acidic environment, and tumor-surrounding astrocyte activation. Temozolomide inhibits GBM growth in brain Hi-Spots, but it is not effective in 2D cultures. Other chemotherapeutics that have proven to be inefficient in GBM treatment display low activity against GBM-derived cells growing in brain Hi-Spots in comparison to their activity against GBM 2D cultures. These findings suggest that GBM/brain Hi-Spots represent a simple system to culture cells derived from brain tumors in an orthotopic environment in vitro and that the system is reliable to test GBM targeting compounds.     

The pathology of sponge orange band disease affecting the Caribbean barrel sponge Xestospongia muta

The aim of this study was to examine sponge orange band (SOB) disease affecting the prominent Caribbean sponge Xestospongia muta. Scanning and transmission electron microscopy revealed that SOB is accompanied by the massive destruction of the pinacoderm. Chlorophyll a content and the main secondary metabolites, tetrahydrofurans, characteristic of X. muta, were significantly lower in bleached than in healthy tissues. Denaturing gradient gel electrophoresis using cyanobacteria-specific 16S rRNA gene primers revealed a distinct shift from the Synechococcus/Prochlorococcus clade of sponge symbionts towards several clades of unspecific cyanobacteria, including lineages associated with coral disease (i.e. Leptolyngbya sp.). Underwater infection experiments were conducted by transplanting bleached cores into healthy individuals, but revealed no signs of SOB development. This study provided no evidence for the involvement of a specific microbial pathogen as an etiologic agent of disease; hence, the cause of SOB disease in X. muta remains unidentified.     

Growth in short term primary cell cultures derived from pregnancy-dependent mammary tumors.

The behavior of cells in primary cultures derived from autonomous and pregnancy-dependent mouse mammary tumors was studied. Despite initial growth both dependent and autonomous mammary tumors produced only short-term primary cultures. Initial plating density had a marked effect on growth with only cultures plated at greater than or equal to 2 X 10(5) cells/cm2 showing any short-termed growth. Time lapse analysis showed that the lack of growth was due to failures of cytokinesis and increased death rate and intermitotic time in cultures plated at less than 2 X 10(5) cells/cm2. Using continuous label autoradiographic techniques, a partial synchronous wave of DNA synthesis was observed with newly plated and restimulated cultures. DNA synthesis reached a peak 24-48 hrs. after plating or restimulation and then dropped to low values for the next few days. Attempts to maintain the initial high rate of DNA synthesis or to induce another round of DNA synthesis by enriched media, increased serum concentrations, or other types of serum and plasma were at best only partially successful. Important hormones necessary for growth of mammary tissue in vivo may be necessary for sustained growth in vitro.     

Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and × collagen and matrix mineralization

In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type X collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hypertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentiation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or X collagen antibodies revealed that type X collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and X collagen, indicating further differentiation of chondrocytes to a new type of 'post-hypertrophic' cell. This cell type, descending from a type X collagen producing chondrocyte, is different from the previously described 'dedifferentiated' or 'modulated' types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type X collagen synthesis. First calcium deposits were detected by alizarine red S staining in type X collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type X collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis.     

Anthranilate sulfonamide hydroxamate TACE inhibitors. Part 1: Structure-based design of novel acetylenic P1' groups

The structure-based design of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1' groups has led to compounds with excellent in vitro potency against TACE and selectivity over MMP-1.     

P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis

Nucleotides are released from cells in response to mechanical stimuli and signal in an autocrine/paracrine manner through cell surface P2 receptors. P2rx7-/- mice exhibit diminished appositional growth of long bones and impaired responses to mechanical loading. We find that calvarial sutures are wider in P2rx7-/- mice. Functional P2X7 receptors are expressed on osteoblasts in situ and in vitro. Activation of P2X7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells. Moreover, osteogenesis is suppressed in calvarial cell cultures from P2rx7-/- mice compared with the wild type. P2X7 receptors couple to production of the potent lipid mediators lysophosphatidic acid (LPA) and prostaglandin E2. Either an LPA receptor antagonist or cyclooxygenase (COX) inhibitors abolish the stimulatory effects of P2X7 receptor activation on osteogenesis. We conclude that P2X7 receptors enhance osteoblast function through a cell-autonomous mechanism. Furthermore, a novel signaling axis links P2X7 receptors to production of LPA and COX metabolites, which in turn stimulate osteogenesis.     

LPS regulation of the immune response: Bacteroides endotoxin induces mitogenic, polyclonal, and antibody responses in classical LPS responsive but not C3H/HeJ mice

Recent studies have suggested that lipopolysaccharide (LPS) derived from gram-negative organisms such as Bacteroides, which are not members of the Enterobacteriaceae, stimulate B cells from the classic LPS-hyporesponsive C3H/HeJ mouse. In the present study, purified, phenol-water-extracted LPS from Bacteroides fragilis ATCC 25285 (B-LPS) was tested for its ability to induce in vivo and in vitro responses in classic LPS-responsive C3H/HeN, LPS-hyporesponsive C3H/HeJ, and (C3H/HeN X C3H/HeJ)F1 hybrid mice. B-LPS induced mitogenic responses in both C3H/HeN and C3H/HeJ spleen cell cultures when cells were cultured under standard conditions, i.e., 8 X 10(5) cells/well. Interestingly, when lower spleen cell numbers were tested with B-LPS, a typical responsive-nonresponsive pattern developed in which good mitogenic responses were induced by B-LPS in C3H/HeN cultures and in which low responses in C3H/HeJ spleen cell cultures were evident. In vivo immunization of mice with B-LPS resulted in high antibody responses in C3H/HeN, intermediate responses in F1, and low responses in C3H/HeJ mice. When purified splenic B cells were incubated with B-LPS, both mitogenic responses and polyclonal immunoglobulin M (IgM) synthesis occurred in C3H/HeN cultures, whereas intermediate responses were noted in F1 cultures and no response was seen in B cell cultures from C3H/HeJ mice. Furthermore, in vitro TNP-B-LPS responses were induced in C3H/HeN spleen cells or purified B cell cultures, and intermediate anti-TNP PFC responses occurred in F1 spleen cells or purified B cell cultures. The toxicity of B-LPS was tested in galactosamine-sensitized mice. The LD50 values for B-LPS in classic LPS-responsive C3H/HeN and C57BL/6J mice were 0.6 microgram and 1.1 microgram, respectively; F1 hybrid mice were approximately 15-fold more resistant, whereas C3H/HeJ mice gave an LD50 of 1650 micrograms. This study shows that phenol-water preparations of B-LPS are biologically active and induce responses in the classic LPS-responsive but not in the LPS-hyporesponsive C3H/HeJ mouse strain.