Synthesis and opioid activity of new fentanyl analogs

Three new fentanyl analogs (compounds 3-4-5) have been synthesized and evaluated for antinociceptive properties using the writhing test. The analgesic property of the active compound, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (compound 4), was tested using the hot plate test in mice. Its opioid agonistic activity was characterized using three isolated tissues: guinea pig ileum, mouse vas deferens, and rabbit vas deferens. Compound 4 was as effective as fentanyl or morphine and it showed less antinociceptive potency than fentanyl but it was more potent than morphine. The duration of the antinociception was similar to that of fentanyl. This compound inhibited the electrically evoked contractions of myenteric plexus-longitudinal muscle strips of guinea pig ileum and of mouse vas deferens but not those of rabbit vas deferens. These effects could be reversed by micro selective antagonists (naloxone and/or CTOP) but not by the delta selective antagonist naltrindole, thus indicating that the compound acted as a micro opioid agonist. Finally, the binding data confirmed that compound 4 had high affinity and selectivity for the micro-receptor.     

Structure of the periplasmic domain of Pseudomonas aeruginosa TolA: evidence for an evolutionary relationship with the TonB transporter protein

The crystal structure of the C-terminal domain III of Pseudomonas aeruginosa TolA has been determined at 1.9 A resolution. The fold is similar to that of the corresponding domain of Escherichia coli TolA, despite the limited amino acid sequence identity of the two proteins (20%). A pattern was discerned that conserves the fold of domain III within the wider TolA family and, moreover, reveals a relationship between TolA domain III and the C-terminal domain of the TonB transporter proteins. We propose that the TolA and TonB C-terminal domains have a common evolutionary origin and are related by means of domain swapping, with interesting mechanistic implications. We have also determined the overall shape of the didomain, domains II + III, of P.aeruginosa TolA by solution X-ray scattering. The molecule is monomeric-its elongated, stalk shape can accommodate the crystal structure of domain III at one end, and an elongated helical bundle within the portion corresponding to domain II. Based on these data, a model for the periplasmic domains of P.aeruginosa TolA is presented that may explain the inferred allosteric properties of members of the TolA family. The mechanisms of TolA-mediated entry of bateriophages in P.aeruginosa and E.coli are likely to be similar.     

Hepatitis C virus core protein leads to immune suppression and liver damage in a transgenic murine model

Hepatitis C virus (HCV) is remarkably efficient in establishing persistent infection, possibly mediated by an impaired immune response to HCV infection. There is compelling evidence that HCV can infect immune cells, such as macrophages, B cells, and T cells. It has been previously reported that HCV core, the first protein expressed during the early phase of viral infection, contains the immunomodulatory function of suppressing host immune responses. This altered function of immune cells caused by HCV infection may explain the ineffective immune response to HCV. To further characterize the immunomodulatory role of HCV core in vivo, we generated transgenic (TG) mice by directing the expression of core protein to T lymphocytes by using the CD2 promoter. T-lymphocyte responses, including the production of gamma interferon and interleukin-2, were significantly diminished in these mice compared to their non-TG littermates. The inhibition of T-lymphocyte responsiveness may be due to the increased susceptibility of peripheral T lymphocytes to Fas-mediated apoptosis. Surprisingly, significant lymphocyte infiltration was observed in the portal tracts of livers isolated from core TG mice, associated with increasing serum alanine aminotransferase levels. Moreover, no intrahepatic lymphocytes or liver damage was found in non-TG littermates and core TG mice bred to Fas-deficient lpr mice. These results suggest that HCV core drives liver injury by increasing Fas-mediated apoptosis and liver infiltration of peripheral T cells.     

Ultrastructure of male sex pheromone glands in abdominal tergites of five Lutzomyia sandfly species (Diptera: Psychodidae)

The fine structure of male sex pheromone disseminating structures on abdominal segments of five species of Lutzomyia sandflies (Diptera: Psychodidae) was analyzed. Scanning electron microscopy showed that in Lutzomyia cruzi [Mem. Inst. Oswaldo Cruz 33 (1938) 349] and Lutzomyia longipalpis [Mem. Inst. Oswaldo Cruz 4 (1912) 84], the disseminating structures are located in pale spots on abdominal tergites IV or III and IV and are morphologically similar, appearing as small round cuticular elevations with a central pore. Observation of abdominal tergites of L. longipalpis pupae showed that the spots, but not the structures, are already present in this stage. In Lutzomyia lenti [Mem. Inst. Oswaldo Cruz 33 (1938) 349] and Lutzomyia carmelinoi [Mem. Inst. Oswaldo Cruz 81 (1986) 323] adult males, the disseminating structures are present on tergal segments V and VI, where pale spots could not be observed, and appear as apple-like elevations with a central pore. In Lutzomyia renei, a single disseminating structure is found at the anterior region of tergal segment VI. Transmission electron microscopy was used to analyze the gland cell fine structure in L. cruzi. Several class III gland cells are located side by side in the fourth abdominal segment. Each epidermal secretory cell contains a small reservoir and a short outlet channel through the cuticle.     

Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing

Heparin-deficient mice, generated by gene targeting of N-deacetylase/N-sulfotransferase-2 (NDST-2), display severe mast cell defects, including an absence of stored mast cell proteases. However, the mechanism behind these observations is not clear. Here we show that NDST-2+/+ bone marrow-derived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparin-like polysaccharide. The corresponding NDST-2-/- cells produced highly sulphated CS only. Carboxypeptidase A (CPA) activity was detected in NDST+/+ cells but was almost absent in the NDST-/- cells, whereas tryptase (mouse mast cell protease 6; mMCP-6) activity and antigen was detected in both cell types. Antigen for the chymase mMCP-5 was detected in NDST-2+/+ cells but not in the heparin-deficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wild-type and NDST-2-/- cells. A approximately 36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a approximately 50 kDa pro-CPA band was present in NDST-2+/+ cells. The NDST-2-/- mast cells contained similar levels of pro-CPA as the wild-type mast cells, but the approximately 36 kDa band was totally absent. This indicates that the processing of pro-CPA to its active form may require the presence of heparin and provides the first insight into a mechanism by which the absence of heparin may cause disturbed secretory granule organisation in mast cells.     

In vitro culture of Cedrela fissilis Vellozo (Meliaceae)

An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments from juvenile origin for axillary shoot proliferation. Shoot proliferation was significantly affected by salt formulation, explant origin and 6-benzyladenine concentration. Maximum multiplication rates (6–7 new plants were produced in the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoog media supplemented with 1.25–5.0 M 6-benzyladenine. Addition of -naphthaleneacetic acid to these media caused significant inhibition on shoot proliferation and growth and stimulated callus formation. High frequency callus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium supplemented with 6-benzyladenine at either 1.25, 2.5 or 5.0 M combined, respectively, with 2.5, 1.25–5.0 or 5.0 M -naphthaleneacetic acid. Rooting was achieved, after 10–12 days, with 87–100% of the node cuttings on half strength Murashige and Skoog medium either without growth regulators or supplemented with 2.5 M indole-3-butyric acid. Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but for further plant development the sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets under ex vitro conditions was 100% after 3 months. The optimized micropropagation and callus culture protocols offer the possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondary metabolism studies.     

The distribution of 3-hydroxy oxylipins in fungi

One of the best-kept secrets by fungi especially yeast is the function of the different shapes and surface structures of their vegetative and sexual cells. They definitely do not produce these shapes (e.g. round, elongated, kidney, needle, hat, saturnoid, etc.) and surfaces (e.g. smooth, rough, hairy, warty, etc.) for our curiosity or to be classified, but surely produce these for their own benefit. This mini-review will show that a large variety of 3-hydroxy oxylipins are widely distributed in the fungal domain and closely associated with these surface ornamentations. In concert with nano-scale surface structures, they probably play a role in cell aggregation as well as spore release from sexual structures such as asci.