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.     

Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.     

Ultrastructural analysis of differentiation in Legionella pneumophila

Legionella pneumophila is an adaptive pathogen that replicates in the intracellular environment of fundamentally divergent hosts (freshwater protozoa and mammalian cells) and is capable of surviving long periods of starvation in water when between hosts. Physiological adaptation to these quite diverse environments seems to be accompanied by morphological changes (Gardu?o et al., p. 82-85, in Marre et al., ed., Legionella, 2001) and conceivably involves developmental differentiation. In following the fine-structural pathway of L. pneumophila through both in vitro and in vivo growth cycles, we have now discovered that this bacterium displays an unprecedented number of morphological forms, as revealed in ultrathin sections and freeze-fracture replicas for transmission electron microscopy. Many of the forms were identified by the obvious ultrastructural properties of their cell envelope, which included changes in the relative opaqueness of membrane leaflets, vesiculation, and/or profuse invagination of the inner membrane. These changes were best documented with image analysis software to obtain intensity tracings of the envelope in cross sections. Also prominent were changes in the distribution of intramembranous particles (clearly revealed in replicas of freeze-fractured specimens) and the formation of cytoplasmic inclusions. Our results confirm that L. pneumophila is a highly pleomorphic bacterium and clarify some early observations suggesting sporogenic differentiation in L. pneumophila. Since morphological changes occurred in a conserved sequence within the growth cycle, our results also provide strong evidence for the existence of a developmental cycle in L. pneumophila that is likely accompanied by profound physiological alterations and stage-specific patterns of gene expression.     

Relationship between long-term resistance to Trypanosoma cruzi and latent infection,examined by antibody production and polymerase chain reaction in mice

Trypanosoma cruzi infections persist for the lifetime of humans and laboratory animals as either latent or pathogenic parasitism. Mice inoculated with a nonpathogenic, attenuated strain (TCC) display resistance against virulent challenge, with a strong control of parasitemia and protection against tissue lesions for more than 12 mo. Three main approaches were used to test whether protection by TCC inocula is based on a latent infection or on a "sterile" immunological memory: curative Benznidazole (Bzl) treatment, serological reactions, and detection of infection by polymerase chain reaction (PCR). If resistance is maintained in the absence of infection, it should not be reduced by Bzl treatment and TCC-inoculated animals should not maintain long-term serological or PCR reactivity. The Bzl treatment after TCC inoculations did not reduce, after periods of up to 420 days, TCC-induced resistance to challenge. But TCC inocula given during Bzl treatment conferred short-term, but not long-term. protection. Maintenance of high antibody levels and protection were better in the virulent Tulahuen (TUL) strain than in the attenuated TCC strain infections, and trypomastigote inocula of either strain were better inducers of antibodies and resistance than epimastigotes. PCR detection of T. cruzi DNA was positive in almost all TUL strain-inoculated animals and negative in immunocompetent animals inoculated with TCC epimastigotes, although high numbers of TCC trypomastigotes produced persistent PCR signals of infection in newborn BALB mice. Thus, 2 polar models were developed, where latent infection by TCC was either demonstrated or excluded. In both, resistance to virulent challenge was maintained during long periods. But late declination of antibody titers (>200 days) and resistance to challenge (>350 days) was observed in animals displaying clearance of all signals of infection.     

Seroprevalence of antibodies to Neospora caninum in dogs from Spain

The prevalence of Neospora caninum antibodies was determined in sera of 139 dogs from Catalonia (northeastern Spain) using the indirect immunofluorescence antibody test (IFAT). Antibodies in the IFAT were found in 17 of 139 dogs (12.2%) with titers ranging from 1:50 to 1: 1,600. Seroprevalence was higher in dogs over 1 yr old compared with dogs younger than 1 yr (P < 0.05). No statistical difference was observed when sex, breed, purpose, or modus vivendi was compared with seropositivity. Most dogs had low antibody titers, which indicated subclinical infection in the area studied. No neosporosis-related disease was reported from any dog, although a German shepherd with an antibody titer of 1:800 showed pododermatitis. All sera were also screened using a commercial direct agglutination test (DAT). The DAT showed a similar specificity but a lower sensitivity when compared with IFAT as a reference technique.     

Generation of amyloid beta protein from a presenilin-1 and betaAPP complex

Presenilin-1 (PS1) is a causative gene in early onset familial Alzheimer's disease (FAD). FAD-linked mutant PS1s significantly increased Abeta40 and Abeta42(43) levels (P < 0.001) and decreased the production of an 11.4 kD (beta-stub) and an 8.7 kD (alpha-stub) carboxyl-terminal fragment of amyloid beta precursor protein (betaAPP-CTFs) (P < 0.01). In the 2% CHAPS extracted lysates, the complex containing the amino-terminal fragment of PS1 (PS1-NTF), the carboxyl-terminal fragments of PS1 (PS1-CTF), and betaAPP-CTFs was identified. Incubation of this isolated complex at pH 6.4 showed the direct generation of Abeta40 and gamma-stub from this complex. This reaction was inhibited by a gamma-secretase inhibitor. The degrading rate of a co-precipitated beta-stub was facilitated under the presence of FAD-linked mutant PS1s. This findings suggest that the direct generation of Abeta from the complex may play an important role in the pathogenesis of Alzheimer's disease.     

The new bilaterally pedicled V-Y advancement flap for face reconstruction

A new bilaterally pedicled V-Y advancement flap based on two subcutaneous pedicles that vascularize the skin island through subdermal plexus lateral bridges is described for face reconstruction. It differs from traditional V-Y advancement flaps in that it does not rely on the classic subcutaneous "vertical" pedicle that is sectioned from top to bottom to improve advancement of the skin island. This technique had predictable results for 12 years in 425 consecutive patients, with infection occurring in 2.8 percent of the cases and complete necrosis in less than 1 percent of the flaps.     

Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration

To test the hypothesis that inhibition of axonal transport is sufficient to cause motor neuron degeneration such as that observed in amyotrophic lateral sclerosis (ALS), we engineered a targeted disruption of the dynein-dynactin complex in postnatal motor neurons of transgenic mice. Dynamitin overexpression was found to disassemble dynactin, a required activator of cytoplasmic dynein, resulting in an inhibition of retrograde axonal transport. Mice overexpressing dynamitin demonstrate a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, and denervation of muscle. Previous transgenic mouse models of ALS have shown abnormalities in microtubule-based axonal transport. In this report, we describe a mouse model that confirms the critical role of disrupted axonal transport in the pathogenesis of motor neuron degenerative disease.