Determination of the absolute configuration of Anisotome irregular diterpenes: application of CD and NMR methods

Several Anisotome diterpene derivatives were synthesized in an attempt to obtain a crystalline compound for X-ray analysis. Although we were unable to obtain a suitable crystal, the absolute configuration of the irregular diterpene skeleton was determined using two other techniques: a circular dichroism (CD) protocol based on a tetraarylporphyrin molecular tweezer that allowed prediction of the absolute stereochemistry on a microscale level, and a method employing differences in NMR shifts from derivatization of the naturally occurring acid 1 with enantiomers of a phenylglycine methyl ester (PGME) chiral anisotropic reagent. The excellent agreement between the CD and NMR methods led to the assignment of a 2S-absolute configuration for anisotomenoic acid 1.     

Cholecystokinin pathways modulate sensations induced by gastric distension in humans

Ingested fat releases CCK, causes gastric relaxation, delays gastric emptying, and limits meal size; however, the mechanistic link among these actions has not been established. Fatty acid release of CCK is chain-length sensitive; dodecanoic acid (C12) induces greater CCK release than decanoic acid (C10). The effect of C12 or C10 on tolerance to subsequent intragastric infusion of liquid was determined in healthy subjects, with and without the CCK(1) receptor antagonist dexloxiglumide. Gastric wall relaxation after either fatty acid was assessed by graded volume distension and by barostat; gastric emptying was measured by gastric aspiration and by a [(13)C]octanoic acid breath technique. C12 released more CCK (mean plasma CCK after vehicle, 4.7 +/- 0.8 pM; C10, 4.8 +/- 0.3 pM; C12, 8 +/- 1.2 pM; P < 0.05 C12 vs. C10 or vehicle) and reduced the volume of water (and of 5 and 25% glucose solutions) delivered at maximum tolerance compared with C10 or vehicle (volume of water tolerated after vehicle, 1,535 +/- 164 ml; C10, 1,335 +/- 160 ml; C12, 842 +/- 103 ml; P < 0.05 C12 vs. C10 or vehicle); this effect was abolished by dexloxiglumide. Intragastric volumes were always similar at the limit of tolerance, and, whereas gastric relaxation occurred to similar degrees after the fatty acids, its duration was longer after C12, which also induced a longer delay in half-gastric emptying [t(1/2)(min) after vehicle, 53 +/- 2; C10, 67 +/- 3; C12, 88 +/- 7; P < 0.05 C12 vs. C10 or vehicle]. In conclusion, ingestion of a CCK-releasing fatty acid reduces the tolerated volume of liquid delivered into the stomach, primarily via a CCK(1) receptor-mediated delay in gastric emptying.     

Expression and functional characterization of SCaMPER: a sphingolipid-modulated calcium channel of cardiomyocytes

Calciumchannels are important in a variety of cellular events including musclecontraction, signaling, proliferation, and apoptosis.Sphingolipids have been recognized as mediators of intracellularcalcium release through their actions on a calcium channel,sphingolipid calcium release-mediating protein of the endoplasmicreticulum (SCaMPER). The current study investigates the expression andfunction of SCaMPER in cardiomyocytes. Northern analyses and RT-PCRcloning and sequencing revealed SCaMPER expression in both human andrat cardiac tissue. Immunofluorescence and Western blot analysesdemonstrated that SCaMPER is abundant in cardiac tissue and islocalized to the sarcotubular junction. This was confirmed by thecolocalization of SCaMPER with dihydropyridine and ryanodine receptorsby confocal microscopy. Purified T tubules were shown to containSCaMPER and immunoelectron micrographs suggested that SCaMPER islocated to the junctional T tubules, but a junctional SR localizationcannot be ruled out. The sphingolipid ligand for SCaMPER,sphingosylphosphorylcholine (SPC), initiated calcium release from thecardiomyocyte SR. Importantly, antisense knockdown of SCaMPER mRNAproduced a substantial reduction of sphingolipid-induced calciumrelease, suggesting that SCaMPER is a potentially important calciumchannel of cardiomyocytes.

     

Structure-Based Phylogenies of the Serine β-Lactamases

The serine -lactamases present a special problem for phylogenetics because they have diverged so much that they fall into three classes that share no detectable sequence homology among themselves. Here we offer a solution to the problem in the form of two phylogenies that are based on a protein structure alignment. In the first, structural alignments were used as a guide for aligning amino acid sequences and in the second, the average root mean square distances between the alpha carbons of the proteins were used to create a pairwise distance matrix from which a neighbor-joining phylogeny was created. From those phylogenies, we show that the Class A and Class D -lactamases are sister taxa and that the divergence of the Class C -lactamases predated the divergence of the Class A and Class D -lactamases.     

Gustatory neurons derived from epibranchial placodes are attracted to, and trophically supported by, taste bud-bearing endoderm in vitro

Taste buds are multicellular receptor organs innervated by the VIIth, IXth, and Xth cranial nerves. In most vertebrates, taste buds differentiate after nerve fibers have reached the lingual epithelium, suggesting that nerves induce taste buds. However, under experimental conditions, taste buds of amphibians develop independently of innervation. Thus, rather than being induced by nerves, the developing taste periphery likely regulates ingrowing nerve fibers. To test this idea, we devised a culture approach using axolotl embryos. Gustatory neurons were generated from cultured epibranchial placodes, and when cultured alone, axon outgrowth was random over 4 days, a time period coincident with axon growth to the periphery in vivo. In contrast, cocultures of placodal neurons with oropharyngeal endoderm (OPE), the normal taste bud-containing target for these neurons, resulted in neurite growth toward the target tissue. Unexpectedly, placodal neurons also grew toward flank ectoderm (FE), which these neurons do not encounter in vivo. To compare further the impact of OPE and FE explants on gustatory neurons, cocultures were extended and examined at 6, 8, and 10 days, when, in vivo, placodal fibers have innervated the epithelium but prior to taste bud formation, when taste buds have differentiated and are innervated, and when the mouth has opened and larvae have begun to feed, respectively. The behavior of placodal axons with respect to target type did not differ between OPE and FE cocultures at 6 days. However, by 8 days, differences in axonal outgrowth were observed with respect to target type, and these differences were enhanced by 10 days in vitro. Most clearly, exuberant placodal fibers grew in 10-day OPE cocultures, and numerous neurites had invaded OPE explants by this time, whereas gustatory neurites were sparse in FE cocultures, and rarely approached and almost never contacted FE explants. Thus, embryonic endoderm destined to give rise to taste buds specifically attracts its innervation early in development, as placodal neurons send out axons. Later, when gustatory axons synapse with differentiated taste buds in vivo, the OPE provides trophic support for cultured gustatory neurons.     

Experimental prediction of the natural evolution of antibiotic resistance

The TEM family of beta-lactamases has evolved to confer resistance to most of the beta-lactam antibiotics, but not to cefepime. To determine whether the TEM beta-lactamases have the potential to evolve cefepime resistance, we evolved the ancestral TEM allele, TEM-1, in vitro and selected for cefepime resistance. After four rounds of mutagenesis and selection for increased cefepime resistance each of eight independent populations reached a level equivalent to clinical resistance. All eight evolved alleles increased the level of cefepime resistance by a factor of at least 32, and the best allele improved by a factor of 512. Sequencing showed that alleles contained from two to six amino acid substitutions, many of which were shared among alleles, and that the best allele contained only three substitutions.     

Non-imprinted Igf2r expression decreases growth and rescues the Tme mutation in mice

In the mouse the insulin-like growth factor receptor type 2 gene (Igf2r) is imprinted and maternally expressed. Igf2r encodes a trans-membrane receptor that transports mannose-6-phosphate tagged proteins and insulin-like growth factor 2 to lysosomes. During development the receptor reduces the amount of insulin-like growth factors and thereby decreases embryonic growth. The dosage of the gene is tightly regulated by genomic imprinting, leaving only the maternal copy of the gene active. Although the function of Igf2r in development is well established, the function of imprinting the gene remains elusive. Gene targeting experiments in mouse have demonstrated that the majority of genes are not sensitive to gene dosage, and mice heterozygous for mutations generally lack phenotypic alterations. To investigate whether reduction of Igf2r gene dosage by genomic imprinting has functional consequences for development we generated a non-imprinted allele (R2Delta). We restored biallelic expression to Igf2r by deleting a critical element for repression of the paternal allele (region 2) in mouse embryonic stem cells. Maternal inheritance of the R2Delta allele has no phenotype; however, paternal inheritance results in biallelic expression of Igf2r, which causes a 20% reduction in weight late in embryonic development that persists into adulthood. Paternal inheritance of the R2Delta allele rescues the lethality of a maternally inherited Igf2r null allele and a maternally inherited Tme (T-associated maternal effect) mutation. These data show that the biological function of imprinting Igf2r is to increase birth weight and they also establish Igf2r as the Tme gene.