Two hybridization events define the population structure of Trypanosoma cruzi

Genetic variation in Trypanosoma cruzi is likely a key determinant in transmission and pathogenesis of Chagas disease. We have examined nine loci as markers for the extant T. cruzi strains. Four distinct alleles were found for each locus, corresponding to the sequence classes present in the homozygous discrete typing units (DTUs) I, IIa, IIb, and IIc. The alleles in DTUs IIa and IIc showed a spectrum of polymorphism ranging from DTU I-like to DTU IIb-like, in addition to DTU-specific sequence variation. DTUs IId and IIe were indistinguishable, showing DTU homozygosity at one locus and heterozygosity with DTU IIb and IIc allelic sequences at eight loci. Recombination between the DTU IIb and IIc alleles is evidenced from mosaic polymorphisms. These data imply that two discrete hybridization events resulted in the formation of the current DTUs. We propose a model in which a fusion between ancestral DTU I and IIb strains gave rise to a heterozygous hybrid that homogenized its genome to become the homozygous progenitor of DTUs IIa and IIc. The second hybridization between DTU IIb and IIc strains that generated DTUs IId and IIe resulted in extensive heterozygosity with subsequent recombination of parental genotypes.     

Yellow Pigments of Aspergillus niger and Aspergillus awamori

Alkaline degradation of Aurasperone A, C₃₂H₂₆O₁₀, gave a binaphthyl (IIa), m.p. 255°C and acetone. (IIa) afforded a tetraacetate (IIb), C₃₂H₃₀O₁₂ m.p. 219°C and a tetramethyl ether (IId), C₂₈H₃₀O₈, m.p. 188°C. These facts along with the NMR spectra of aurasperone A and (IIb) confirm that aurasperone A is a dimeric 2-methyl-5-hydroxy-6,8-dimethoxy-4H-naphtho[2,3-b]pyran-4-one with asymmetric C-C linkage (7-10′ or 9-10′). The ether (IId) is not identical with 1,1′ ,3,3′ ?6,6′ ,8,8′-octamethoxy-4,4′-binaphthyl. Thus, it follows that (IId) is a 2,4′-binaphthyl and hence aurasperone A is 2,2′-dimethyl-5,5′- dihydroxy-6,6′,8,8′-tetrahydroxy-7,10′-bi[4H-naphtho[2,3-b]pyran-4-one] (I).     

Synthesis of β-Tubanol Methylether

β-Tubanol methylether (IIb) was obtained by the Grignard reaction from methyl magnesium iodide and 5-methoxy-coumarin in boiling benzene. The preparation of 2-methyl-4-(2′-hydroxy-6′-methoxyphenyl) -buten-3-ol-2 (VI) and 2,2-dimethyl-7-methoxy-chromene-3 (IId) were also described.     

in vitro inhibition of the measles virus by novel ring-expanded ('fat') nucleoside analogues containing the imidazo[4,5-e]diazepine ring system

The synthesis and in vitro anti-measles virus (anti-MV) activity of a class of ring-expanded ('fat') nucleoside analogues (1-4) containing the title heterocyclic ring system are reported. The target compounds were synthesized by base-catalyzed condensations of 4,5-dicarboxylic acid esters of the appropriately substituted imidazole-1-ribosides with suitably substituted guanidine derivatives. Compounds were screened for anti-MV activity in African green monkey kidney cells (CV-1), employing ribavirin as the control standard. While the parent compound 1 itself failed to show any significant antiviral activity against MV, its analogues containing hydrophobic substituents at the 2-position (2) or the 6-position (4) showed promising antiviral activity at submicromolar or micromolar concentration levels with no apparent toxicity to the host cell line. Both compounds showed higher anti-MV activity than the control drug ribavirin.     

Growth and Muscle Defects in Mice Lacking Adult Myosin Heavy Chain Genes

The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying null mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both null strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb null mutants are generally milder than in the MyHC-IId/x null strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for null expression of the two genes. Most striking is that while both null strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb null mice has significantly reduced ability to generate force while IId null mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.     

Skeletal muscle adaptations in response to voluntary wheel running in myosin heavy chain null mice.

10.1152/ japplphysiol.00832.2001.-To examine the effects of gene inactivation on the plasticity of skeletal muscle, mice null for a specific myosin heavy chain (MHC) isoform were subjected to a voluntary wheel-running paradigm. Despite reduced running performance compared with nontransgenic C57BL/6 mice (NTG), both MHC IIb and MHC IId/x null animals exhibited increased muscle fiber size and muscle oxidative capacity with wheel running. In the MHC IIb null animals, there was no significant change in the percentage of muscle fibers expressing a particular MHC isoform with voluntary wheel running at any time point. In MHC IId/x null mice, wheel running produced a significant increase in the percentage of fibers expressing MHC IIa and MHC I and a significant decrease in the percentage of fibers expressing MHC IIb. Muscle pathology was not affected by wheel running for either MHC null strain. In summary, despite their phenotypes, MHC null mice do engage in voluntary wheel running. Although this wheel-running activity is lessened compared with NTG, there is evidence of distinct patterns of muscle adaptation in both null strains.     

Degradation of substituted indoles by an indole-degrading methanogenic consortium.

Degradation of indole by an indole-degrading methanogenic consortium enriched from sewage sludge proceeded through a two-step hydroxylation pathway yielding oxindole and isatin. The ability of this consortium to hydroxylate and subsequently degrade substituted indoles was investigated. Of the substituted indoles tested, the consortium was able to transform or degrade 3-methylindole and 3-indolyl acetate. Oxindole, 3-methyloxindole, and indoxyl were identified as metabolites of indole, 3-methylindole, and 3-indolyl acetate degradation, respectively. Isatin (indole-2,3-dione) was produced as an intermediate when the consortium was amended with oxindole, providing evidence that degradation of indole proceeded through successive hydroxylation of the 2- and 3-positions prior to ring cleavage between the C-2 and C-3 atoms on the pyrrole ring of indole. The presence of a methyl group (-CH3) at either the 1- or 2-position of indole inhibited the initial hydroxylation reaction. The substituted indole, 3-methylindole, was hydroxylated in the 2-position but not in the 3-position and could not be further metabolized through the oxindole-isatin pathway. Indoxyl (indole-3-one), the deacetylated product of 3-indolyl acetate, was not hydroxylated in the 2-position and thus was not further metabolized by the consortium. When an H atom or electron-donating group (i.e., -CH3) was present at the 3-position, hydroxylation proceeded at the 2-position, but the presence of electron-withdrawing substituent groups (i.e., -OH or -COOH) at the 3-position inhibited hydroxylation.     

Postnatal myosin heavy chain isoform expression in normal mice and mice null for IIb or IId myosin heavy chains

The patterns of myosin heavy chain (MyHC) isoform expression in the embryo and in the adult mouse are reasonably well characterized and quite distinct. However, little is known about the transition between these two states, which involves major decreases and increases in the expression of several MyHC genes. In the present study, the expression of seven sarcomeric MyHCs was analyzed in the hindlimb muscles of wild-type mice and in mice null for the MyHC IIb or IId/x genes at several time points from 1 day of postnatal life (dpn) to 20 dpn. In early postnatal life, the developmental isoforms (embryonic and perinatal) comprise >90% of the total MyHC expression, while three adult fast isoforms (IIa, IIb, and IId) comprise <1% of the total MyHC protein. However, between 5 and 20 dpn their expression increases to comprise >90% of the total MyHC. Expression of each of the three adult fast isoforms occurs in a spatially and temporally distinct manner. We also show that alpha MyHC, which is almost exclusively expressed in the heart, is expressed in scattered fibers in all hindlimb muscles during postnatal development. Surprisingly, the timing and localization of expression of the MyHC isoforms is unchanged in IIb and IId/x null mice, although the magnitude of expression is altered for some isoforms. Together these data provide a comprehensive overview of the postnatal expression pattern of the sarcomeric MyHC isoforms in the mouse hindlimb.     

Degradation of substituted indoles by an indole-degrading methanogenic consortium.

Degradation of indole by an indole-degrading methanogenic consortium enriched from sewage sludge proceeded through a two-step hydroxylation pathway yielding oxindole and isatin. The ability of this consortium to hydroxylate and subsequently degrade substituted indoles was investigated. Of the substituted indoles tested, the consortium was able to transform or degrade 3-methylindole and 3-indolyl acetate. Oxindole, 3-methyloxindole, and indoxyl were identified as metabolites of indole, 3-methylindole, and 3-indolyl acetate degradation, respectively. Isatin (indole-2,3-dione) was produced as an intermediate when the consortium was amended with oxindole, providing evidence that degradation of indole proceeded through successive hydroxylation of the 2- and 3-positions prior to ring cleavage between the C-2 and C-3 atoms on the pyrrole ring of indole. The presence of a methyl group (-CH3) at either the 1- or 2-position of indole inhibited the initial hydroxylation reaction. The substituted indole, 3-methylindole, was hydroxylated in the 2-position but not in the 3-position and could not be further metabolized through the oxindole-isatin pathway. Indoxyl (indole-3-one), the deacetylated product of 3-indolyl acetate, was not hydroxylated in the 2-position and thus was not further metabolized by the consortium. When an H atom or electron-donating group (i.e., -CH3) was present at the 3-position, hydroxylation proceeded at the 2-position, but the presence of electron-withdrawing substituent groups (i.e., -OH or -COOH) at the 3-position inhibited hydroxylation.     

Spatial and temporal changes in myosin heavy chain gene expression in skeletal muscle development

Seven myosin heavy chains (MyHC) are expressed in mammalian skeletal muscle in spatially and temporally regulated patterns. The timing, distribution, and quantitation of MyHC expression during development and early postnatal life of the mouse are reported here. The three adult fast MyHC RNAs (IIa, IIb, and IId/x) are expressed in the mouse embryo and each mRNA has a distinct temporal and spatial distribution. In situ hybridization analysis demonstrates expression of IIb mRNA by 14.5 dpc, which proceeds developmentally in a rostral to caudal pattern. IId/x and IIa mRNAs are detectable 2 days later. Ribonuclease protection assays demonstrate that the three adult fast genes are expressed at approximately equal levels relative to each other in the embryo but at quite low levels relative to the two developmental isoforms, embryonic and perinatal. Just after birth major changes in the relative proportions of different MyHC RNAs and protein occur. In all cases, RNA expression and protein expression appear coincident. The changes in MyHC RNA and protein expression are distinct in different muscles and are restricted in some cases to particular regions of the muscle and do not always reflect their distribution in the adult.     

Synthesis and structure-activity relationship (SAR) study of 4-azabenzoxazole analogues as H3 antagonists

The synthesis and SAR of a novel series of 4-azabenzoxazole histamine H(3) antagonists is described. Introduction of substituted phenyl, pyridyl and fused heterocyclic groups to the 6-position of the 4-azabenzoxazole core gave a series of compounds with good H(3) antagonist activity in both ex vivo and in vivo assays.     

The effect of triacyl-sn-glycerol structure on the metabolism of chylomicrons and triacylglycerol-rich emulsions in the rat

A systematic study was undertaken to observe the effects of dietary (dioleoyl) triacyl-sn-glycerol structure on chylomicron composition and metabolism. First studied was a series of 1,2-dioleoyl-3-(saturated)acyl-sn-glycerols, where the fatty acid esterified at the 3-position was varied from 14 to 24 carbons. Next a series of 1,3-dioleoyl-2-acyl glycerols was studied, with various fatty acids esterified at the glycerol 2-position. These stereospecific triacyl-sn-glycerols were fed to donor rats and lymph chylomicrons were isolated, analyzed, and reinjected into recipient rats to study their disappearance from plasma and delivery to tissues. As shown by their compositions, chylomicrons obtained after feeding triacylglycerols containing all sn-3 fatty acid of chain length greater than 20 carbons were under-represented, possibly due to poorer digestion by lipases, or poorer absorption by the intestine. The 18-carbon saturated chain fatty acid (stearic acid) was equally well represented in chylomicrons whether in the 2- or 3-position of the fed triacylglycerol. The presence of increased amounts of long-chain saturated fatty acids in donor chylomicron triacylglycerols affected the metabolism of chylomicrons injected into the bloodstream of recipient rats. In particular the rate of removal of labeled cholesteryl esters, tracing removal of the partially degraded chylomicron remnants was slowed by the saturated chains, with palmitic acid and the 20-carbon fatty acid, arachidic acid, showing the most severe effects. There were clear differences in the removal from plasma of injected lymph chylomicrons derived from fed triacylglycerols containing stearic acid in either the 2- or 3-position, with evidence for remnants from the symmetrical triacylglycerols being less rapidly removed from the circulating blood. This effect was investigated further by injected model emulsions of chylomicrons, where the 2-position was substituted with saturated or transunsaturated acyl chains. Quantitation of removal from the blood stream of these model lipoproteins confirmed that a saturated or transunsaturated long chain fatty acid at the 2-position of the emulsion triacylglycerols slowed remnant removal from the blood. In some cases, with both lymph chylomicron and with emulsions, the lipolytic step mediated by lipoprotein lipase was also slowed.     

Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing

Thioether-linked 3-mercaptopropionic acid derivatives of 17beta-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17beta-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.     

Parallel solid-phase synthesis of disubstituted (5-biphenyltetrazolyl) hydantoins and thiohydantoins targeting the growth hormone secretagogue receptor

An efficient solid-phase protocol for the synthesis of substituted (5-biphenyltetrazolyl)-hydantoins and -thiohydantoins has been developed. Suzuki cross-coupling between resin-bound 2-(tetrazol-5-yl)-phenylborinane and 4-bromobenzaldehyde gave the corresponding tetrazolylbiphenyl aldehyde. Subsequent reductive amination using amino acid esters gave the pivotal resin bound amino acid esters which were transformed to hydantoins or thiohydantoins via two routes: (i) treatment with isocyanates or isothiocyanates or (ii) successive treatment with triphosgene and primary amines. Using molecular modeling, we were able to jump from L-692,429, a well known non-peptidyl growth hormone secretagogue (GHS), to biphenyltetrazolyl hydantoins, obtaining compounds with IC(50) values below 600 nM after two iterative cycles only.     

Design, synthesis, and pharmacological evaluation of some 2-[4-morpholino]-3-aryl-5-substituted thiophenes as novel anti-inflammatory agents: generation of a novel anti-inflammatory pharmacophore

Compounds incorporating a thiophene moiety, a pi excess five membered heterocycle, have attracted a great deal of research interest owing to the therapeutic utility of the template as useful drug molecular scaffolding. Recently we reported the anti-inflammatory activity profile exhibited by two thiophene analogs, AP84 and AP82 in acute and chronic models of inflammation. The good activity profile exhibited by AP84, a 3-(substituted aryl)-2-(4-morpholino)-5-heteroaryl substituted analog of thiophene, in the formalin induced rat paw edema chronic model as compared to a weak activity in acute carrageenin induced rat paw edema, and the slightly better protection exhibited in the acute model by AP82 (27%), the 5-aroyl analog provided an impetus for a proper exploration of their structural types. In this paper we report the synthesis and pharmacological evaluation of some novel, 2-(4-morpholino)-3-(substituted aryl)-5-substituted thiophenes, as possible anti-inflammatory leads. The 3-(4-chlorophenyl)-2-(4-morpholino) thiophene analogs AP49, AP158, and AP88 provided a protection of 20%, 23%, and 20%, respectively, when screened for anti-inflammatory activity in carrageenin induced rat paw edema, an acute in vivo model, comparable to that of AP82, at a dose level of 100mg/kg body weight p.o. compared to ibuprofen as standard. The replacement of the 3-(4-chlorophenyl) moiety with the 3-phenyl moiety gave rise to AP50 (30%), AP159 (38%), AP27 (0%), and AP92 (38%), with three analogs being more active in the acute model. Alteration of the group para to the phenyl ring at third position, from chloro, to methyl mercapto gave rise to the 3-(4-methylmercapto-phenyl) analogs AP54 (20%), AP160 (0%), and AP73 (52%), with only one analog appearing to be better than AP82. These results indicate that 4-methane sulfonyl aroyl group at 5-position and other substituents of different quadrants of Craig plot on the phenyl moiety at the third position could lead to more potent candidates. However, alteration of aroyl to substituted pyridyl at 5-position with a phenyl group at the third position as in AP26 gave rise to much better protection (66%) again reinforcing the importance of the heteroaryl ring at the fifth position and implying its utility in the composition of a novel pharmacophore for designing better trisubstituted thiophenes as anti-inflammatory agents.     

Structure-dependent inhibition of bladder and pancreatic cancer cell growth by 2-substituted glycyrrhetinic and ursolic acid derivatives

Derivatives of oleanolic acid, ursolic acid and glycyrrhetinic acid substituted with electron-withdrawing groups at the 2-position in the A-ring which also contains a 1-en-3-one structure are potent inhibitors of cancer cell growth. In this study, we have compared the effects of several 2-substituted analogs of triterpenoid acid methyl esters derived from ursolic and glycyrrhetinic acid on proliferation of KU7 and 253JB-V bladder and Panc-1 and Panc-28 pancreatic cancer cells. The results show that the 2-cyano and 2-trifluoromethyl derivatives were the most active compounds. The glycyrrhetinic acid derivatives with the rearranged C-ring containing the 9(11)-en-12-one structure were generally more active than the corresponding 12-en-11-one isomers. However, differences in growth inhibitory IC(50) values were highly variable and dependent on the 2-substituent (CN vs CF(3)) and cancer cell context.     

Structure and anti-complementary activity of pectic polysaccharides isolated from the root of Angelica acutiloba Kitagawa

Four pectic polysaccharides (AR-2IIa-IId) with anti-complementary activity have been isolated from a hot-water extract of the root of Angelica acutiloba Kitagawa. Each of these polysaccharides contained a large proportion of GalA together with neutral sugars consisting mainly of Rha, Ara, and Gal. Digestion with endo-alpha-(1----4)-polygalacturonase indicated that AR-2IIa-IIc each contained a large proportion of enzyme-sensitive polygalacturonan regions, and that AR-2IId contained a large proportion of enzyme-resistant regions. When AR-2IId was de-esterified, it became sensitive to the enzyme. These polysaccharides also contained small proportions of enzyme-resistant regions (PG-1) which were rich in neutral sugars. Methylation analysis and base-catalysed beta-elimination studies suggested that each PG-1 contained a rhamnogalacturonan moiety in which 2,4-disubstituted Rha was attached to 4-substituted GalA through position 2 of Rha. Carboxyl-reduction and methyl- and de-esterification of these polysaccharides modulated their anti-complementary activities. Digestion with endo-alpha-(1----4)-polygalacturonase decreased the activities of AR-2IIa and -2IIb, but not those of AR-2IIc and -2IId. Although PG-1 fractions from AR-2IIa-IIc were more active than the original polysaccharides, oligogalacturonide fragments obtained by enzymic digestion had weak or negligible activity. AR-2IIa-IIc expressed their anti-complementary activities mainly via the classical pathway, but AR-2IId and each PG-1 expressed their activities via both the classical and alternative pathways.     

Trypanosoma cruzi 5S rRNA arrays define five groups and indicate the geographic origins of an ancestor of the heterozygous hybrids

Isolates of the etiological agent of Chagas disease, Trypanosoma cruzi, have been subdivided into six subgroups referred to as discrete typing units. The subgroups are related through two distinct hybridisation events: representatives of homozygous discrete typing units I and IIb fused to form discrete typing units IIa and IIc, whose homozygous genotypes have features of both ancestral types; a second fusion between strains of homozygous discrete typing units IIb and IIc created the heterozygous hybrid strains discrete typing units IId and IIe. The intergenic region of the tandemly repeated 5S rRNA array displays four variant sequence classes, allowing the discrimination of five discrete typing units. The genome project reference strain, CL Brener, is a hybrid discrete typing unit IIe strain that contains both discrete typing unit IIb and IIc classes of 5S rRNA repeats in distinct arrays present on different chromosomes. The CL Brener discrete typing unit IIb-type array contains approximately 193 repeated units, of which about one-third contain a 129 bp sequence that replaces a majority of the 5S rRNA sequence. The 129 bp 'invader' sequence was detected within the arrays of all hybrid discrete typing unit IId and IIe strains and in a subset of discrete typing unit IIb strains. This array invader replaces the internal promoter elements conserved in 5S rRNA. The discrete typing unit IIb Esmeraldo strain contains approximately 135 repeats and shows a region of homology to the array invader in the 5' flank of the array, but no evidence of the invading sequence element within the array. A survey of additional discrete typing unit IIb strains revealed a split within the subgroup, in which some strains contained invaded arrays and others were homogeneous for the 5S rRNA. The putative discrete typing unit IIb ancestor of the hybrid discrete typing units IId and IIe more closely resembles the extant Bolivian/Chilean IIb isolates than the Brazilian IIb isolates based on the correlation with the array invader.     

Design and Synthesis of 2′-Deoxy-2′-[(1,2,3)Triazol-1-Yl]Uridines Using Click Chemistry Approach

A series of novel nucleosides bearing a 1,2,3-triazole moiety at the 2′-position of the sugar moiety has been synthesized starting from 2′-azidouridine and using the copper (I)-catalyzed Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition reaction. The reactions proceeded in overall yield of 52–82% and gave almost exclusively the 1,4-disubstituted 1,2,3-triazoles. The 2′-azidouridine was synthesized from uridine in two steps, and reacted with a variety of differently substituted alkynes to give the desired 2′-triazole-substituted uridine derivatives.