Synthesis and biological activity of selective pipecolic acid-based TNF-alpha converting enzyme (TACE) inhibitors

A series of novel, selective TNF-alpha converting enzyme inhibitors based on 4-hydroxy and 5-hydroxy pipecolate hydroxamic acid scaffolds is described. The potency and selectivity of TACE inhibition is dramatically influenced by the nature of the sulfonamide group which interacts with the S1' site of the enzyme. Substituted 4-benzyloxybenzenesulfonamides exhibit excellent TACE potency with >100x selectivity over inhibition of matrix metalloprotease-1 (MMP-1). Alkyl substituents on the ortho position of the benzyl ether moiety give the most potent inhibition of TNF-alpha release in LPS-treated human whole blood.     

Plasma kinetics of VLDL and HDL apoC-I in normolipidemic and hypertriglyceridemic subjects

ApoC-I has several different lipid-regulating functions including, inhibition of receptor-mediated uptake of plasma triglyceride-rich lipoproteins, inhibition of cholesteryl ester transfer activity, and mediation of tissue fatty acid uptake. Since little is known about the rate of production and catabolism of plasma apoC-I in humans, the present study was undertaken to determine the plasma kinetics of VLDL and HDL apoC-I using a primed constant (12 h) intravenous infusion of deuterium-labeled leucine. Data were obtained for 14 subjects: normolipidemics (NL, n = 4), hypertriglyceridemics (HTG, n = 4) and combined hyperlipidemics (CHL, n = 6). Plasma VLDL triglyceride (TG) levels were 0.59 +/- 0.03, 4.32 +/- 0.77 (P < 0.01 vs. NL), and 2.20 +/- 0.39 mmol/l (P < 0.01 vs. NL), and plasma LDL cholesterol (LDL-C) levels were 2.34 +/- 0.22, 2.48 +/- 0.26, and 5.35 +/- 0.48 mmol/l (P < 0.01 vs. NL), respectively. HTG and CHL had significantly (P < 0.05) increased levels of total plasma apoC-I (12.5 +/- 1.2 and 12.4 +/- 1.3 mg/dl, respectively) versus NL (7.9 +/- 0.6 mg/dl), due to significantly (P < 0.01) elevated levels of VLDL apoC-I (5.8 +/- 0.8 and 4.5 +/- 0.8 vs. 0.3 +/- 0.1 mg/dl). HTG and CHL also had increased rates of VLDL apoC-I transport (i.e., production) versus NL: 2.29 +/- 0.34 and 3.04 +/- 0.53 versus 0.24 +/- 0.11 mg/kg.day (P < 0.01), with no significant change in VLDL apoC-I residence times (RT): 1.16 +/- 0.12 versus 0.69 +/- 0.06 versus 0.74 +/- 0.17. Although HDL apoC-I concentrations were not significantly lower in HTG and CHL versus NL, HDL apoC-I rates of transport were inversely related to plasma and VLDL-TG levels (r = -0.63 and -0.62, respectively, P < 0.05). Our results demonstrate that increased levels of plasma and VLDL apoC-I in hypertriglyceridemic subjects (with or without elevated LDL-C levels) are associated with increased levels of plasma VLDL apoC-I production.     

Urea-selective concentrating defect in transgenic mice lacking urea transporter UT-B.

Urea transporter UT-B has been proposed to be the major urea transporter in erythrocytes and kidney-descending vasa recta. The mouse UT-B cDNA was isolated and encodes a 384-amino acid urea-transporting glycoprotein expressed in kidney, spleen, brain, ureter, and urinary bladder. The mouse UT-B gene was analyzed, and UT-B knockout mice were generated by targeted gene deletion of exons 3-6. The survival and growth of UT-B knockout mice were not different from wild-type littermates. Urea permeability was 45-fold lower in erythrocytes from knockout mice than from those in wild-type mice. Daily urine output was 1.5-fold greater in UT-B- deficient mice (p < 0.01), and urine osmolality (U(osm)) was lower (1532 +/- 71 versus 2056 +/- 83 mosM/kg H(2)O, mean +/- S.E., p < 0.001). After 24 h of water deprivation, U(osm) (in mosM/kg H(2)O) was 2403 +/- 38 in UT-B null mice and 3438 +/- 98 in wild-type mice (p < 0.001). Plasma urea concentration (P(urea)) was 30% higher, and urine urea concentration (U(urea)) was 35% lower in knockout mice than in wild-type mice, resulting in a much lower U(urea)/P(urea) ratio (61 +/- 5 versus 124 +/- 9, p < 0.001). Thus, the capacity to concentrate urea in the urine is more severely impaired than the capacity to concentrate other solutes. Together with data showing a disproportionate reduction in the concentration of urea compared with salt in homogenized renal inner medullas of UT-B null mice, these data define a novel "urea-selective" urinary concentrating defect in UT-B null mice. The UT-B null mice generated for these studies should also be useful in establishing the role of facilitated urea transport in extrarenal organs expressing UT-B.     

Ly-49s3 is a promiscuous activating rat NK cell receptor for nonclassical MHC class I-encoded target ligands

Previous studies of the rapid rejection of MHC-disparate lymphocytes in rats, named allogeneic lymphocyte cytotoxicity, have indicated that rat NK cells express activating receptors for nonclassical MHC class I allodeterminants from the RT1-C/E/M region. Using an expression cloning system that identifies activating receptors associated with the transmembrane adapter molecule DAP12, we have cloned a novel rat Ly-49 receptor that we have termed Ly-49 stimulatory receptor 3 (Ly-49s3). A newly generated anti-Ly-49s3 Ab, mAb DAR13, identified subpopulations of resting and IL-2-activated NK cells, but not T or B lymphocytes. Depletion of Ly-49s3-expressing NK cells drastically reduced alloreactivity in vitro, indicating that this subpopulation is responsible for a major part of the observed NK alloreactivity. DAR13-mediated blockade of Ly-49s3 inhibited killing of MHC-congenic target cells from the av1, n, lv1, and c haplotypes, but not from the u or b haplotypes. A putative ligand was mapped to the nonclassical MHC class I region (RT1-C/E/M) using intra-MHC recombinant strains. Relative numbers of Ly-49s3(+) NK cells were reduced, and surface levels of Ly-49s3 were lower, in MHC congenic strains expressing the putative Ly-49s3 ligand(s). In conclusion, we have identified a novel Ly-49 receptor that triggers rat NK cell-mediated responses.     

Cyanogenesis in glucosinolate-producing plants: Carica papaya and Carica quercifolia

(R)-2-(beta-D-Glucopyranosyloxy)-2-phenylacetonitrile (prunasin) was isolated from Carica papaya L. and C. quercifolia (A. St.-Hil.) Hieron. (syn. C. hastata Brign.). Earlier reported presence of cyclopentanoid cyanohydrin glycosides in C. papaya could not be confirmed, and no cyclopentanoid amino acids could be detected in extracts of C. papaya and C. quercifolia. Conversion of [2,3,4,5,6-3H]phenylalanine into tritiated prunasin was demonstrated in both species. On the other hand, when the plants were administered [2-14C]-2-(2'cyclopentenyl)glycine, extracted, and the extracts hydrolyzed with beta-glucosidase (Helix pomatia), formation of labelled cyanide was not observed. The absence of cyclopentanoids, which are typical for the Passifloraceae, and the inability of Carica species to utilize 2-(2'-cyclopentenyl)glycine as a precursor of cyanogenic glycosides are in agreement with the relative phylogenetic position of the Caricaceae and the Passifloraceae. Carica species are thus rare examples of taxa in which glucosinolates and cyanogenic glycosides co-occur, both types of natural products being derived from the same amino acid, phenylalanine.     

Effect of leaf surface waxes on leaf colonization by Pantoea agglomerans and Clavibacter michiganensis

To evaluate the influence of leaf cuticular waxes on bacterial colonization of leaves, bacterial colonization patterns were examined on four glossy maize (Zea mays L.) mutants that were altered in their cuticular wax biosynthesis. Mutant gl3 was indistinguishable from the wild-type maize in its ability to foster colonization by the two bacterial species, Pantoea agglomerans and Clavibacter michiganensis subsp. nebraskensis. In contrast, the other three mutants supported the development of populations that significantly differed in size from those on the wild type. Mutant gl5 gl20 supported smaller populations of P. agglomerans, but not C. michiganensis, while mutant gl1 supported larger populations of C. michiganensis but not P. agglomerans. Mutant gl4 supported larger populations of both bacterial species. The exceptional ability of mutant gl4 to support bacterial colonization was hypothesized to result from the lower density of the crystalline waxes on gl4 than on the wild type, because a reduced crystal density could promote capillary water movement and water trapping among the wax crystals. This hypothesis was supported by the demonstration that the mechanical introduction of gaps among the wax crystals of the wild-type leaves resulted in the establishment of larger P. agglomerans populations on the altered leaves. These results provide the first direct evidence that leaf surface waxes affect bacterial leaf colonization at various stages of colonization and in a bacterial species-dependent manner.     

Adjuvant immunotherapy for solid tumors: from promise to clinical application

Although surgery remains the mainstay for the treatment of most solid tumors, investigators are seeking complementary therapies to eradicate microscopic disease, which causes tumor relapse even after an apparently complete surgical excision. Although adjuvant chemotherapy has achieved some significant results, the control of minimal residual disease is still a challenge for clinicians. Among novel therapeutic approaches, immunotherapy holds promise. This anticancer strategy aims at triggering a highly specific endogenous killing machine against tumor cells. Recent progress in tumor immunology has improved our understanding of host-immune system interactions. In particular, new technologies have fostered the identification of potentially immunogenic tumor antigens that can be used as suitable targets for immune effector cells. After observing immunotherapy-mediated clinical responses in patients with metastatic disease, investigators have started evaluating this anticancer modality in the adjuvant setting. Here, we review the immunological strategies so far explored in humans and report worldwide results following the clinical application of adjuvant immunotherapy for solid tumors.     

Mitochondrial RNase P RNAs in ascomycete fungi: lineage-specific variations in RNA secondary structure

The RNA subunit of mitochondrial RNase P (mtP-RNA) is encoded by a mitochondrial gene (rnpB) in several ascomycete fungi and in the protists Reclinomonas americana and Nephroselmis olivacea. By searching for universally conserved structural elements, we have identified previously unknown rnpB genes in the mitochondrial DNAs (mtDNAs) of two fission yeasts, Schizosaccharomyces pombe and Schizosaccharomyces octosporus; in the budding yeast Pichia canadensis; and in the archiascomycete Taphrina deformans. The expression of mtP-RNAs of the predicted size was experimentally confirmed in the two fission yeasts, and their precise 5' and 3' ends were determined by sequencing of cDNAs generated from circularized mtP-RNAs. Comparative RNA secondary structure modeling shows that in contrast to mtP-RNAs of the two protists R. americana and N. olivacea, those of ascomycete fungi all have highly reduced secondary structures. In certain budding yeasts, such as Saccharomycopsis fibuligera, we find only the two most conserved pairings, P1 and P4. A P18 pairing is conserved in Saccharomyces cerevisiae and its close relatives, whereas nearly half of the minimum bacterial consensus structure is retained in the RNAs of fission yeasts, Aspergillus nidulans and Taphrina deformans. The evolutionary implications of the reduction of mtP-RNA structures in ascomycetes will be discussed.     

Synthesis of 2'-spiro ribo- and arabinonucleosides

Four conformationally restricted bicyclic 2'-spiro nucleosides were synthesised and incorporated into oligonucleotides. These spiro nucleotides induced decreased duplex thermostabilities.     

Hemispheric dissociation in access to the human semantic system

Patient studies suggest that speech and environmental sounds are differentially processed by the left and right hemispheres. Here, using functional imaging in normal subjects, we compared semantic processing of spoken words to equivalent processing of environmental sounds, after controlling for low-level perceptual differences. Words enhanced activation in left anterior and posterior superior temporal regions, while environmental sounds enhanced activation in a right posterior superior temporal region. This left/right dissociation was unchanged by different attentional/working memory contexts, but it was specific to tasks requiring semantic analysis. While semantic processing involves widely distributed networks in both hemispheres, our results support the hypothesis of a dual access route specific for verbal and nonverbal material, respectively.