Discovery and optimization of orally active cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors

The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.     

When invasion biology meets taxonomy: <Emphasis Type="Italic">Clavelina oblonga</Emphasis> (Ascidiacea) is an old invader in the Mediterranean Sea

Taxonomic issues often confound the study of invasive species, which sometimes are unrecognized as introduced in newly colonized areas. Clavelina oblonga Herdman, 1880 is an abundant ascidian species along the southeastern coast of the United States and the Caribbean Sea. It was introduced into the eastern Atlantic and Brazil decades ago. In the Mediterranean Sea, a similar species had been described as C. phlegraea Salfi 1929 and reported from southern Italy and Corsica. In the last few years a species of Clavelina has proliferated in the embayments of the Ebro Delta (NW Mediterranean), a zone of active bivalve culture industry where it has smothered mussel spat, leading to economic loss. We here report the morphological and genetic identity of this species, synonymizing the Atlantic C. oblonga and the Mediterranean C. phlegraea (the latter therefore is a synonym of the former). Thus, C. oblonga has existed in the Mediterranean for over 80 years, but was known under a different name. We also found this species in natural habitats in the Iberian Atlantic coast close to the Strait of Gibraltar, raising concerns about an ongoing expansion. In order to obtain information relevant for management, we monitored growth, reproductive cycles and settlement patterns of this ascidian on bivalve cultures in the Ebro Delta. Its biological cycles were markedly seasonal, with peak abundance and reproduction during the warmest months, followed by regression during the cold season. The settlement period was short, mostly concentrated in a single month each year. Avoidance of mussel and oyster seeding during late summer and early autumn can readily reduce the damage caused by this species.     

Effects of the new adiponectin paralogous protein CTRP-3 and of LPS on cytokine release from monocytes of patients with type 2 diabetes mellitus

Aims/Hypothesis: It was the aim to investigate the hypothesis that the new C1q/TNF-family member CTRP-3 (C1q/TNF-related protein-3) acts anti-inflammatory in human monocytes from healthy controls and patients with type 2 diabetes mellitus (T2D). Methods: Monocytes were isolated from 20 healthy controls and 30 patients with T2D. IL-6 and TNF concentrations were measured by ELISA. CTRP-3 was expressed in insect cells and used for stimulation experiments. Results: Basal IL-6 and TNF were not different in control and in T2D monocytes. LPS-stimulation (1 μg/ml) significantly (p < 0.001) increased IL-6 and TNF in the supernatants of control and in T2D monocytes to a similar extent. CTRP-3 (1 μg/ml) significantly (p = 0.03) inhibited LPS-induced IL-6 in control monocytes but not in T2D monocytes. TNF upon co-stimulation with LPS and CTRP-3 was significantly (p = 0.012) lower in control than in T2D monocytes. LPS-induced TNF concentration was significantly and positively correlated with serum total cholesterol and LDL cholesterol in T2D patients. Conclusions: CTRP-3 inhibits LPS-induced IL-6 and TNF release. This anti-inflammatory effect is lost in T2D. Serum cholesterol concentration affects the pro-inflammatory potential of LPS to induce TNF release from T2D monocytes in the presence or absence of CTRP-3. CTRP-3 might partly account for the pro-inflammatory state in T2D.     

Unusual viral ligand with alternative interactions is presented by HLA-Cw4 in human respiratory syncytial virus-infected cells

Short viral antigens bound to human major histocompatibility complex (HLA) class I molecules are presented on infected cells. Vaccine development frequently relies on synthetic peptides to identify optimal HLA class I ligands. However, when natural peptides are analyzed, more complex mixtures are found. By immunoproteomics analysis, we identify in this study a physiologically processed HLA ligand derived from the human respiratory syncytial virus matrix protein that is very different from what was expected from studies with synthetic peptides. This natural HLA-Cw4 class I ligand uses alternative interactions to the anchor motifs previously described for its presenting HLA-Cw4 class I molecule. Finally, this octameric peptide shares its C-terminal core with the H-2D(b) nonamer ligand previously identified in the mouse model. These data have implications for the identification of antiviral cytotoxic T lymphocyte responses and for vaccine development.     

Expression, localization, and functional activity of TL1A, a novel Th1-polarizing cytokine in inflammatory bowel disease

TL1A is a novel TNF-like factor that acts as a costimulator of IFN-gamma secretion through binding to the death domain-containing receptor, DR3. The aim of this study was to test the hypothesis that TL1A may play an important role in inflammatory bowel disease (IBD) by functioning as a Th1-polarizing cytokine. The expression, cellular localization, and functional activity of TL1A and DR3 were studied in intestinal tissue specimens as well as isolated lamina propria mononuclear cells from IBD patients and controls. TL1A mRNA and protein expression was up-regulated in IBD, particularly in involved areas of Crohn's disease (CD; p < 0.03 vs control). TL1A production was localized to the intestinal lamina propria in macrophages and CD4(+) and CD8(+) lymphocytes from CD patients as well as in plasma cells from ulcerative colitis patients. The amount of TL1A protein and the number of TL1A-positive cells correlated with the severity of inflammation, most significantly in CD. Increased numbers of immunoreactive DR3-positive T lymphocytes were detected in the intestinal lamina propria from IBD patients. Addition of recombinant human TL1A to cultures of PHA-stimulated lamina propria mononuclear from CD patients significantly augmented IFN-gamma production by 4-fold, whereas a minimal effect was observed in control patients. Our study provides evidence for the first time that the novel cytokine TL1A may play an important role in a Th1-mediated disease such as CD.     

Early detection in saliva of polypeptides associated to isoproterenol-induced mouse parotid hypertrophy

Chronic administration of isoproterenol (IPR) results in a marked hypertrophy and in the induction of a group of putative proline-rich polypeptides in the mouse parotid glands. Some of these polypeptides (pps C-G) have been considered as molecular markers of the parotid gland enlargement. Given the secretory character of polypeptides C-G, the polypeptide composition of mouse saliva was used to monitor the IPR-induced salivary gland hypertrophy. Whole saliva was collected after an oral administration of pilocarpine (PIL). Under those conditions, PIL provoked a massive salivary secretion both in normal control mice and during the whole course of the IPR-induced gland enlargement. Striking changes in the polypeptide composition of saliva obtained from chronically IPR-stimulated animals were observed. Those changes consisted basically in the appearance and progressive increase in concentration of parotid polypeptides C-G and in the progressive diminution in concentration of a couple of normal salivary polypeptides (polypeptides A-B). The appearance of new polypeptides in saliva could be established unequivocally within the 24 h following the trophic adrenergic stimulation. On the other hand, salivary polypeptides induced in response to a single administration of IPR could be demonstrated as late as 7-9 days after the stimulation. Accordingly, detection of parotid polypeptides C-G in PIL-produced saliva obtained from IPR-stimulated mice has proved to be a highly advantageous method to evaluate salivary gland hypertrophy both at very early stages after the trophic stimulation and late after the occurrence of the trophic episode.     

In vitro and in vivo assays of 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives against Trypanosoma cruzi

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 micro g/ml for macrophages and a few of them maintained this cytotoxicity even at 10 microg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1microg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.     

Mouse novel Ly9: a new member of the expanding CD150 (SLAM) family of leukocyte cell-surface receptors

Human CS1, also known as novel Ly9, 19A24, or CRACC, is a member of the immunoglobulin gene superfamily (IgSF) expressed on natural killer cells and other leukocytes. Here we describe the cloning of the mouse homologue of this gene. The mouse novel Ly9 gene is shown to encode a transmembrane protein composed of two extracellular immunoglobulin-like domains, a transmembrane region and an 88-amino acid cytoplasmic domain. Mouse novel Ly9 is structurally similar to the extracellular domains of CD84 and CD229 (Ly9). Both mouse and human novel Ly9 genes mapped close to the CD229gene in a region where other members of the CD150 family have also been mapped, and analysis of their genomic sequences showed that they have an identical intron/exon organization. Northern blot analysis revealed that the expression of mouse and human novel Ly9 was predominantly restricted to hematopoietic tissues, with the exception of testis. Here we show that SAP (SH2D1A), an adapter protein responsible for the X-linked lymphoproliferative disease, binds to the phosphorylated cytoplasmic tail of human but not mouse novel Ly9. Taken together, these data indicate that mouse novel Ly9 is a new member of the expanding CD150 family of cell surface receptors.     

Population structure in the endangered Blanca Cacereña bovine breed demonstrated by RAPD analyses

RAPD analyses have been used to determine the genetic diversity and the population structure of the endangered Blanca Cacere?a bovine breed. Genetic variability was evaluated on the basis of 1048 loci produced by 71 primers. RAPD produced a number of polymorphic loci (30.44%), and it has been proved to be a useful method for evaluating polymorphisms in this breed. The dendrograms based on simililarity indexes and on Nei's genetic distances between 60 animals and the value of genetic differentiation among subpopulations (F(ST)) showed a clear population substructure defined by herds and a scarce genetic flow among herds. Analysis of molecular variance (AMOVA) showed that 32.4% of the total variance was due to differences among herds and confirmed the clustering found. The results of the present study allow us to plan more adequate mating in order to maintain the genetic diversity and to improve the efficiency of conservation for the Blanca Cacere?a bovine breed.