Synthetic studies in the series of polyene macrolide antibiotics. 6. Synthesis of methyl-2,4,6-trideoxy-2,4-dimethyl-alpha-altro- hexopyranoside, the C33-C38 fragment of amphotericin B

The synthesis of methyl 2,4,6-trideoxy-2,4-dimethyl-alpha-L-altro-hexopyranoside, having the configuration of the C33-C38 fragment of amphotericin B, is described.     

Profiling C6-C3 and C6-C1 phenolic metabolites in Cocos nucifera

This paper reports the detection and identification of phenolic metabolites (C6-C3 and C6-C1 compounds) in Cocos nucifera. An HPLC/UV system was used to analyze the soluble and wall-associated phenolics in mesocarp and leaf tissues of C. nucifera. Alkaline hydrolysis of the cell wall material of the mesocarpic and leaf tissues yielded 4-hydroxybenzoic acid as the major phenolic compound. Other phenolic acids identified were ferulic acid, 4-coumaric acid, 4-hydroxybenzaldehyde and vanillic acid. No significant qualitative differences in composition were observed between leaf and mesocarp, but there were quantitative variations in the metabolite levels.     

Synthetic studies of polyene macrolide antibiotics. 2. Synthesis of methyl-4,6-dideoxy-2,3-isopropylidene-4-C-methyl-alpha-L- mannopyranoside

The synthesis of methyl 4,6-dideoxy-2,3-O-isopropylidene-4-C-methyl-alpha-L-mannopyranoside, a key intermediate on the preparation of C33-C38 fragment of amphotericin B, from L-rhamnose is described.     

Synthetic studies of polyene macrolide antibiotics. 3. Hydrogenation of methyl-6-deoxy-2,3-O-isopropylidene-4-methylene- alpha-L-lyxo-hexopyranoside

Experimental conditions of hydrogenation of the title compound are found (excess of Raney catalyst in benzene solution, 5 degrees C, 2h) to afford methyl 4,6-dideoxy-2,3-O-isopropylidene-4-C-methyl-alpha-L-mannopyranoside, the key intermediate in the synthesis of C33-C38 fragment of amphotericin B, with the yield as high as 57%.     

Chemical activation of C1-C2 spinal neurons modulates intercostal and phrenic nerve activity in rats

Chemical activation of upper cervical spinal neurons modulates activity of thoracic respiratory interneurons in rats. The aim of the present study was to examine the effects of chemical activation of C(1)-C(2) spinal neurons on thoracic spinal respiratory motor outflows. Electroneurograms of left phrenic (n = 23) and intercostal nerves (ICNs, n = 93) between T(3) and T(8) spinal segments were recorded from 36 decerebrated, vagotomized, paralyzed, and ventilated male rats. To activate upper cervical spinal neurons, glutamate pledgets (1 M, 1 min) were placed on the dorsal surface of the C(1)-C(2) spinal cord. Glutamate on C(1)-C(2) increased ICN tonic activity in 56/59 (95%) ICNs. The average maximal tonic activity of ICN was increased by 174% (n = 59). After spinal transection at rostral C(1), glutamate on C(1)-C(2) still increased ICN tonic activity in 33/35 ICNs. However, the effects of C(1)-C(2) glutamate on ICN phasic activity were highly variable, with observations of augmentation or suppression of both inspiratory and expiratory discharge. C(1)-C(2) glutamate augmented the average amplitude of phrenic burst by 20%, whereas the increases in amplitude of ICN inspiratory activity, when they occurred, averaged 120%. The burst rate of phrenic nerve discharge was decreased from 34.2 +/- 1.6 to 26.3 +/- 2.0 (mean +/- SE) breaths/min during C(1)-C(2) glutamate. These data suggested that upper cervical propriospinal neurons might play a role in descending modulation of thoracic respiratory and nonrespiratory motor activity.     

Function and stability of human transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187

The current studies have investigated the role of three disulfide bonds of human transcobalamin II (TC II), a plasma transporter of cobalamin (Cbl; vitamin B12), in its function and stability. When translated in vitro in the presence or absence of microsomal vesicles, TC II constructs with a single substitution, C3S or C249S, demonstrated synthesis of a stable functional protein. However, TC II synthesized in the presence of microsomal vesicles using constructs with a single (C98S, C147S, C187S, C291S), double (C3/147/S, C98/147/S) or triple (C3/98/147/S) substitution was unstable. In the absence of microsomal vesicles, the percentage of binding to Cbl-Sepharose matrix by TC II expressed by constructs C3S, C3/147/S, C98/147/S, or C3/98/147/S was 100, 49, 52, and 35%, respectively. Upon their reductive alkylation, the binding of TC II expressed by these constructs was reduced to approximately 25-30%. TC II constructs C3S or C249S, when expressed in TC II-deficient fibroblasts, produced a stable functional protein, but those expressed by constructs C147S, C187S, C291S, C3/147/S, C98/147/S, or C3/98/147/S were rapidly degraded. The intracellular degradation of TC II expressed by these constructs was inhibited by lactacystin or MG-132 but not by the lysosomal degradation inhibitors ammonium chloride or chloroquine. These studies suggest that optimal binding of Cbl by human TC II is supported by disulfide bonds C98-C291 and C147-C187 and that their disruption results in loss of Cbl binding and their rapid degradation by the proteasomal machinery.     

Characteristics of a strain of C. albicans resistant to polyene antibiotics]

It was found that a resistant strain R2 of C. albicans obtained as a result of passages on media containing increasing concentrations of amphotericin B differed from the initial strain by its lower pathogenicity. Treatment of the infection caused by the resistant strain on modeling of candidiasis in mice was not successful. The decrease in the average life span of the mice infected with the resistant strain R2 and treated with amphotericin B was lower than that in the control animals and such indices of the disease as the levels of the kidney dissemination and the cell vegetation even increased under the effect of amphotericin B. The results of the study suggest that the resistant strain R2 of C. albicans depend on amphotericin B in the host. The data obtained emphasize the necessity of determinining the antibiotic sensitivity of C. albicans strains isolated from patients.     

Electrophysiological properties of heteromeric TRPV4-C1 channels

We previously reported that TRPV4 and TRPC1 can co-assemble to form heteromeric TRPV4-C1 channels [12]. In the present study, we characterized some basic electrophysiological properties of heteromeric TRPV4-C1 channels. 4α-Phorbol 12,13-didecanoate (4α-PDD, a TRPV4 agonist) activated a single channel current in HEK293 cells co-expressing TRPV4 and TRPC1. The activity of the channels was abrogated by a TRPC1-targeting blocking antibody T1E3. Conductance of the channels was ~95pS for outward currents and ~83pS for inward currents. The channels with similar conductance were also recorded in cells expressing TRPV4-C1 concatamers, in which assembled channels were expected to be mostly 2V4:2C1. Fluorescence Resonance Energy Transfer (FRET) experiments confirmed the formation of a protein complex with 2V4:2C1 stoichiometry while suggesting an unlikeliness of 3V4:1C1 or 1V4:3C1 stoichiometry. Monovalent cation permeability profiles were compared between heteromeric TRPV4-C1 and homomeric TRPV4 channels. For heteromeric TRPV4-C1 channels, their permeation profile was found to fit to Eisenman sequence VI, indicative of a strong field strength cation binding site, whereas for homomeric TRPV4 channels, their permeation profile corresponded to Eisenman sequence IV for a weak field strength binding site. Compared to homomeric TRPV4 channels, heteromeric TRPV4-C1 channels were slightly more permeable to Ca2+ and had a reduced sensitivity to extracellular Ca2+ inhibition. In summary, we found that, when TRPV4 and TRPC1 were co-expressed in HEK293 cells, the predominate assembly type was 2V4:2C1. The heteromeric TRPV4-C1 channels display distinct electrophysiological properties different from those of homomeric TRPV4 channels.     

Intrapericardiac injections of algogenic chemicals excite primate C1-C2 spinothalamic tract neurons

Extracellular potentials of 38 C1-C2 spinothalamic tract (STT) neurons in anesthetized monkeys (Macaca fascicularis) were examined for responses to intrapericardiac injections of an algogenic chemical mixture (adenosine, 10(-3) M; bradykinin, prostaglandin E(2), serotonin, histamine, each 10(-5) M). Chemical stimulation of cardiac/pericardiac receptors increased activity of 21 cells, decreased activity of 5 cells, and did not change activity of 12 cells. Cells excited by chemical stimuli received input from noxious mechanical stimulation of somatic fields; most receptive fields included the neck, inferior jaw, or head areas. Nerve ablations in 11 cells excited by intrapericardiac chemicals showed that cardiac input activated by algogenic chemicals traveled primarily in vagal afferent fibers to C1-C2 segments; phrenic or cardiopulmonary sympathetic inputs were predominant in 2 of 11 cells. These results supported the concept that activation of cardiac vagal afferents might lead to the production of referred pain sensation in somatic fields innervated from high cervical segments.     

C4 isoform of NADP-malate dehydrogenase. cDNA cloning and expression in leaves of C4, C3, and C3-C4 intermediate species of Flaveria.

In C4 plants of the NADP-malic enzyme type, an abundant, mesophyll cell-localized NADP-malate dehydrogenase (MDH) acts to convert oxaloacetate, the initial product of carbon fixation, to malate before it is shuttled to the bundle sheath. Since NADP-MDH has different but important roles in leaves of C3 and C4 plants, we have cloned and characterized a nearly full-length cDNA encoding NADP-MDH from Flaveria trinervia (C4) to permit comparative structure/expression studies within the genus flaveria. The dicot genus Flaveria includes C3-C4 intermediate species, as well as C3 and C4 species. We show that the previously noted differences in NADP-MDH activity levels among C3, C4, and C3-C4 Flaveria species are in part due to interspecific differences in mRNA accumulation. We also show that the NADP-MDH gene appears to be present as a single copy among different Flaveria species, suggesting that a pre-existing gene has been reregulated during the evolution from C3 to C4 plants to accommodate the abundance and localization requirements of the C4 cycle.     

Morphological aspects and biomechanical properties of the vertebroaxial joint (C2-C3).

The C2-C3 intervertebral joint must be regarded as a transitional area situated between the upper cervical spine where most rotation of the neck and little flexion and extension occur and the lower cervical spaces where chiefly motion in the sagittal plane and also somewhat rotation take place. Under normal circumstances the range of flexion-extension reaches 11 degrees, slighter than below (19.5 degrees at C5-C6); on the opposite, the range of rotation attains 7 degrees; less than above but much more than below (0 degrees at C5-C6). The motion in the sagittal and coronal planes is relatively poor because of the location of Penning's motor-axis of C2 which runs far from the vertebral body and the lowness of the intervertebral disc. However, the rotation of C2 with respect to C3 is fair by the peculiar inclination of the articular facets which slope sagittally but also coronally and trace a sphere whereupon C2 may move around its motor-centre in any plane. When C2-C3 is surgically fixed by bone graft, the lack of motion is completed by a "compensatory movement" in the upper cervical spaces and especially at the atlantooccipital joint for flexion-extension. In the same way, C2-C3 may improve its mobility especially in the sagittal plane when the inferior partner is blocked by surgical or arthritic fusion.     

Analysis of the Drosophila betaPS subunit indicates that regulation of integrin activity is a primal function of the C8-C9 loop

Integrin-ligand interactions can be influenced by the sequence in a disulfide-bridged loop between the 8th and 9th beta subunit cysteines. Previous experiments are consistent with C8-C9 loop residues being involved in direct ligand-integrin interactions and/or being important in heterodimer regulation. In betaPS from Drosophila melanogaster and three other dipterans, the C8-C9 loop consists of only two amino acids, and exists in two forms that arise by differential splicing of exon 4. In these species, the betaPS4A isoform has an acidic residue in the first loop position (C8+1), with an alanine or proline in the corresponding position of betaPS4B. Mutations in both isoforms (in combination with alphaPS2) can reduce cell spreading during normal growth, but function is generally restored under conditions that enhance integrin activation. Replacement of the betaPS4A acidic residue with a basic lysine has relatively modest effects on integrin function. Spread cells bearing C8-C9 mutations tend to become less elongated, with reduced frequencies of actin stress fibers. The results indicate that even a minimal, two-residue C8-C9 loop contains structural information that can differentially regulate integrin activity and/or integrin signaling, and that this regulation does not rely on direct molecular interactions involving the variable C8+1 side chains.     

Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin

Cardiac myosin-binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin-binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2 (5-30μM, in a buffer containing in mM: 180 KCl, 1 MgCl(2), 1 EDTA, 1 DTT, 20 imidazole, at pH 7.4) with F-actin (5μM) for 30min and examined negatively-stained samples of the solution by electron microscopy (EM). Examination of EM images revealed that C0-C2 bound to F-actin to form long helically-ordered complexes. Fourier transforms indicated that C0-C2 binds with the helical periodicity of actin with strong 1st and 6th layer lines. The results provide direct evidence that the N-terminus of cMyBP-C can bind to F-actin in a periodic complex. This interaction of cMyBP-C with F-actin supports the possibility that binding of cMyBP-C to F-actin may play a role in the regulation of cardiac contraction.     

The functional significance of C3-C4 intermediate traits in Heliotropium L. (Boraginaceae): gas exchange perspectives

We demonstrate for the first time the presence of species exhibiting C3-C4 intermediacy in Heliotropium (sensu lato), a genus with over 100 C3 and 150 C4 species. CO2 compensation points (Gamma) and photosynthetic water-use efficiencies (WUEs) were intermediate between C3 and C4 values in three species of Heliotropium: Heliotropium convolvulaceum (Gamma = 20 micromol CO2 mol(-1) air), Heliotropium racemosum (Gamma = 22 micromol mol(-1)) and Heliotropium greggii (Gamma = 17 micromol mol(-1)). Heliotropium procumbens may also be a weak C3-C4 intermediate based on a slight reduction in Gamma (48.5 micromol CO2 mol(-1)) compared to C3Heliotropium species (52-60 micromol mol(-1)). The intermediate species H. convolvulaceum, H. greggii and H. racemosum exhibited over 50% enhancement of net CO2 assimilation rates at low CO2 levels (200-300 micromol mol(-1)); however, no significant differences in stomatal conductance were observed between the C3 and C3-C4 species. We also assessed the response of Gamma to variation in O2 concentration for these species. Heliotropium convolvulaceum, H. greggii and H. racemosum exhibited similar responses of Gamma to O2 with response slopes that were intermediate between the responses of C3 and C4 species below 210 mmol O2 mol(-1) air. The presence of multiple species displaying C3-C4 intermediate traits indicates that Heliotropium could be a valuable new model for studying the evolutionary transition from C3 to C4 photosynthesis.     

Synthetic studies in the series of polyene macrolide antibiotics. 4. Synthesis of methyl-2,4,6-trideoxy-3-0-benzoyl-2,4-di-C-methyl- alpha-L-talo-hexopyranoside and 2,4,6-trideoxy-2,4-di-C-methyl-L- galactitol, the stereoisomers of the fragment C33-C38 of amphotericin B

Synthesis of methyl 2,4,6-trideoxy-3-O-benzoyl-2,4-di-C-methyl-alpha-L-talohexopyranoside and 2,4,6-trideoxy-2,4-di-C-methyl-L-galactitol, stereoisomers of the C33-C38 fragment of amphothericin B, is described.     

Total synthesis and biological evaluation of potent analogues of dictyostatin: modification of the C2-C6 dienoate region

By exploiting a Still-Gennari olefination of a common C11-C26 aldehyde with a C4-C10 or C1-C10 beta-ketophosphonate, three modified C2-C6 region analogues of the 22-membered macrolide dictyostatin were synthesised and evaluated in vitro for growth inhibition against a range of human cancer cell lines, including the Taxol-resistant NCI/ADR-Res cell line. 6-Desmethyldictyostatin and 2,3-dihydrodictyostatin displayed potent (low nanomolar) antiproliferative activity, intermediate between dictyostatin and discodermolide, while 2,3,4,5-tetrahydrodictyostatin showed activity comparable to discodermolide. As with dictyostatin, these simplified analogues act through a mechanism of microtubule stabilisation, G2/M arrest and apoptosis.     

Immunological characteristics of PEP carboxylase from leaves of C3-, C4- and C3-C4 intermediate species of Alternanthera--comparison with selected C3- and C4- plants

Immunological cross-reactivity of phosphoenolpyruvate carboxylase (PEPC) in leaf extracts of C3-, C4- and C3-C4 intermediate species of Alternanthera (along with a few other C3- and C4- plants) was studied using anti-PEPC antibodies raised against PEPC of Amaranthus hypochondriacus (belonging to the same family as that of Alternanthera, namely Amaranthaceae). Antibodies were also raised in rabbits against the purified PEPC from Zea mays (C4- monocot-Poaceae) as well as Alternanthera pungens (C4- dicot-Amaranthaceae). Monospecificity of PEPC-antiserum was confirmed by immunoprecipitation. Amount of PEPC protein in leaf extracts of A. hypochondriacus could be quantified by single radial immunodiffusion. Cros- reactivity of PEPC in leaf extracts from selected C3-, C4-, and C3-C4 intermediate species (including those of Alternanthera) was examined using Ouchterlony double diffusion and Western blots. Anti-PEPC antiserum raised against A. hypochondriacus enzyme showed high cross-reactivity with PEPC in leaf extracts of A. hypochondriacus or Amaranthus viridis or Alternanthera pungens (all C4 dicots), but limited cross-reactivity with that of Zea mays, Sorghum or Pennisetum (all C4 monocots). Interestingly, PEPC in leaf extracts of Alternanthera tenella, A. ficoides, Parthenium hysterophorus (C3-C4 intermediates) exhibited stronger cross-reactivity (with anti-serum raised against PEPC from Amaranthus hypochondriacus) than that of Pisum sativum, Commelina benghalensis, Altenanthera sessilis (C3 plants). Further studies on cross-reactivities of PEPC in leaf extracts of these plants with anti-PEPC antisera raised against PEPC from leaves of Zea mays or Alternanthera pungens confirmed two points--(i) PEPC of C3-C4 intermediate is distinct from C3 species and intermediate between those of C3- and C4-species; and (ii) PEPC of C4-dicots was closer to that of C3-species or C3-C4 intermediates (dicots) than to that of C4-monocots.     

Synthesis of Southern (C1'-C11') and Eastern (C8-C18) Fragments of Pamamycin-607, an Aerial Mycelium-inducing Substance of Streptomyces alboniger

Synthesis of the southern C1'-C11' and eastern C8-C18 fragments of pamamycin-607, an aerial mycelium-inducing substance of Streptomyces alboniger, was achieved. The southern fragment was synthesized by using the Evans aldol reaction and cis-selective iodoetherification as the key steps in a 9.6% overall yield (7 steps). The eastern fragment was constructed via the Julia coupling reaction and cis-selective iodoetherification in a 3.0% overall yield (8 steps from the known epoxide).     

Regulation of PKC alpha activity by C1-C2 domain interactions

In this study, the role of interdomain interactions involving the C1 and C2 domains in the mechanism of activation of PKC was investigated. Using an in vitro assay containing only purified recombinant proteins and the phorbol ester, 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA), but lacking lipids, it was found that PKC alpha bound specifically, and with high affinity, to a alpha C1A-C1B fusion protein of the same isozyme. The alpha C1A-C1B domain also potently activated the isozyme in a phorbol ester- and diacylglycerol-dependent manner. The level of this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that alpha C1A-C1B bound to a peptide containing the C2 domain of PKC alpha. The alpha C1A-C1B domain also activated conventional PKC beta I, -beta II, and -gamma isoforms, but not novel PKC delta or -epsilon. PKC delta and -epsilon were each activated by their own C1 domains, whereas PKC alpha, -beta I, -beta II, or -gamma activities were unaffected by the C1 domain of PKC delta and only slightly activated by that of PKC epsilon. PKC zeta activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKC alpha results from the dissociation of intra-molecular interactions between the alpha C1A-C1B domain and the C2 domain. Furthermore, it is shown that PKC alpha forms dimers via inter-molecular interactions between the C1 and C2 domains of two neighboring molecules. These mechanisms may also apply for the activation of the other conventional and novel PKC isozymes.