Identification of Muscarinic Receptors in Rat Cerebral Cortical Microvessels

Microvessels isolated from rat cerebral cortex consist mainly of capillaries (greater than 85%). Fresh, intact microvessel preparations have been analyzed by radioligand binding techniques for muscarinic receptors. Scatchard analysis of specific quinuclidinyl benzilate (QNB) binding indicates that microvessels possess a large number of muscarinic sites (914 fmol/mg protein) of high affinity (KD = 0.034 nM). The association and dissociation rate constants (0.37 min-1 nM-1 and 0.0067 min-1, respectively) yield an equilibrium KD of 0.018 nM. Displacement of [3H]QNB by muscarinic ligands and control substances is typical of muscarinic receptors. The results indicate that cerebral microvessels possess a large population of muscarinic receptors.     

Development of a Novel Trap for the Collection of?Black Flies of the Simulium ochraceum?Complex

Background

Human landing collections are currently the standard method for collecting onchocerciasis vectors in Africa and Latin America. As part of the efforts to develop a trap to replace human landing collections for the monitoring and surveillance of onchocerciasis transmission, comprehensive evaluations of several trap types were conducted to assess their ability to collect Simulium ochraceum sensu lato, one of the principal vectors of Onchocerca volvulus in Latin America.

Methodology/Principal Findings

Diverse trap designs with numerous modifications and bait variations were evaluated for their abilities to collect S. Ochraceum s.l. females. These traps targeted mostly host seeking flies. A novel trap dubbed the “Esperanza window trap” showed particular promise over other designs. When baited with CO₂ and BG-lure (a synthetic blend of human odor components) a pair of Esperanza window traps collected numbers of S. Ochraceum s.l. females similar to those collected by a team of vector collectors.

Conclusions/Significance

The Esperanza window trap, when baited with chemical lures and CO₂ can be used to collect epidemiologically significant numbers of Simulium ochraceum s.l., potentially serving as a replacement for human landing collections for evaluation of the transmission of O. volvulus.     

Vaccination of Stage IV patients with allogeneic IL-4- or IL-2-gene-transduced melanoma cells generates functional antibodies against vaccinating and autologous melanoma cells

The antibody (Ab) response to allogeneic Me14932 and autologous melanoma cells was analyzed in 13 Stage IV (AJCC) melanoma patients immunized with Me14932 cells transduced with the IL-4 (Me14932/IL-4) ( n=10) or IL-2 (Me14932/IL-2) ( n=3) gene. No Ab response was observed before the 4th vaccination. Among 8 patients that received four vaccinations, 3/5 patients vaccinated with Me14932/IL-4 cells developed Ab (IgG and/or IgM) to Me14932 ( n=3) and to autologous ( n=2) melanoma cells, and 2/3 patients vaccinated with Me14932/IL-2 cells developed Ab (IgG) to Me14932, but not to autologous melanoma cells. Further, among these 5 responding patients, circulating Ab against the HLA-A3 allele, expressed only on vaccinating cells, were identified in the immune sera of 4 patients immunized with Me14932/IL-4 ( n=2) or Me14932/IL-2 ( n=2) cells. These sera mediated antibody-dependent cell cytotoxicity (ADCC) of Me14932 cells, and a direct correlation ( r=0.85; P=0.03) between intensity of staining (IgG) and extent of lysis was found. Immune serum of one of these patients also induced ADCC of autologous melanoma cells, and serum from another patient mediated complement cytotoxicity of Me14932, but not of autologous melanoma cells. Thus, Abs against vaccinating and autologous melanoma cells were generated in 62% of patients after four vaccinations with cytokine-transduced melanoma cells. These findings demonstrate that the identification and titration of alloreactive Ab helps to monitor the extent of immunization against cellular vaccines, while the induction of Ab reactive to antigens shared between vaccinating and autologous melanoma cells may contribute to their therapeutic efficacy.     

Effect of adenosylhomocysteine and other analog thioethers on a prokaryotic tRNA (guanine-7)-methyltransferase

S-Adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases, and several thioethers structurally related to SAH, have been tested as potential inhibitors of tRNA (guanine-7)-methyltransferase from Salmonella typhimurium. The tested compounds are l-, d-, dl-S-adenosylhomocysteine, S-adenosylcysteine, methylthioadenosine, butylthioadenosine, thioethanoladenosine, isobutylthioadenosine, S-inosylhomocysteine, and methylthioinosine. Among them the highest inhibitory activity has been shown by SAH (K_i = 8 μM), whereas butylthioadenosine, isobutylthioadenosine, and thioethanoladenosine are almost inactive as inhibitors. The other compounds inhibit the enzyme with K_i values ranging between 400 and 800 μm. From these data it is possible to evaluate the importance of the -NH₂ and -COOH groups of the substrate in the binding to the enzyme molecule, as well as other features such as the chirality at the α-carbon atom and the length of the hydrocarbon chain connecting the -NH₂ and -COOH groups to the aromatic ring of adenosine. The aminic group of the adenosine is also critical, because S-inosylhornocysteine and methylthioinosine are poorer inhibitors in comparison with SAH and methylthioadenosine.     

Viability, maturation and embryo development in vitro of immature porcine and ovine oocytes vitrified in different devices

This study was designed to evaluate the effects of vitrification on immature porcine and ovine oocytes, collected at a slaughterhouse, by performing vitrification in devices with different volumes. Viability was evaluated both before and after vitrification and maturation. Immediately after warming, the percentage of viable pig oocytes was 81% regardless the type of device, while in the control (after oocyte selection) was 95%. The viability of matured pig oocytes after warming, vitrified in beveled edge open straws (BES) was 6%, in small-open-pulled-straw (SOPS) was 17% and in cryotop was 4%, while the viability of the control group was 86%. The viability and maturation results were similar with all devices. Embryo development (ED) was observed in fresh porcine oocytes with 15% 2-8 cell embryos, 7% morulae and 3% blastocysts, and non-embryo cleavage was observed in warmed oocytes. The viability of sheep oocytes immediately after warming averaged 90% in all devices, while that of the control (after oocyte selection) averaged 95%. The viability of warmed oocytes after maturation was: BES 21%, SOPS 30%, cryotop 21% and control group 86%; while maturation values were 11, 21, 34 and 70%, respectively. After vitrification, the highest ED was achieved with ovine oocytes vitrified in SOPS, with 17% morulae development and it was the only device in which blastocysts developed. A direct relationship was observed between viability and actin filament integrity in both species.     

Heat shock proteins: biological functions and clinical application as personalized vaccines for human cancer

Heat shock proteins (HSPs) are a large family of proteins with different molecular weights and different intracellular localizations. These proteins undertake crucial functions in maintaining cell homeostasis, and therefore they have been conserved during evolution. Hsp70 and Grp94/gp96, due to their peptide chaperone capacity and their ability to actively interact with professional antigen-presenting cells (APCs), are also endowed with crucial immunological functions. The immunological properties of these proteins and their implications for vaccine in human cancer will be discussed. Immunological and clinical data of phase I/II studies in melanoma and colorectal cancer patients will be reviewed.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Generation of variability by in vivo recombination of halves of a (beta/alpha)8 barrel protein

Similar to what has been achieved with nucleic acids, directed evolution of proteins would be greatly facilitated by the availability of large libraries and efficient selection methods. So far, host cell transformation efficiency has been a bottleneck, practically limiting libraries to sizes less than 10(9). One way to circumvent this problem has been implemented with antibody systems, where contribution to the binding site is provided by two different polypeptides (light and heavy chains). The central concept is the construction of binary systems in which the gene from the two chains are separated by a cre-lox recombinase recognition site, packaged in a phage, and subsequently introduced, by multiple infection, into a recombinase expressing cell [Sblattero D, Bradbury A. Nat Biotechnol 2000;18(1):75-80]. Here, we describe the development of a system which applies the same concept to a single-domain enzyme, the cytoplasmic (beta/alpha)8 barrel protein phosphoribosyl anthranilate isomerase (PRAI) from E. coli. For that purpose, we identified the site at which a loop containing the recognition sequence for cre-lox recombinase could be inserted yielding a functional enzyme. We evaluated the effect of this insertion on the capability of the engineered gene to complement a trp F-E. coli strain and the efficiency of the system to recover the original sequence from an abundance of non-functional mutant genes.     

Neuroendocrine aspects of placenta and pregnancy

Placenta plays a central role in the regulation of physiological mechanisms of pregnancy, and in particular is the organ of communication between mother and fetus. This action is also related to its ability to produce hormones, growth factors and cytokines during the progression of pregnancy, and in response to stimuli such as stress and inflammation/infection. In the last years the understanding of the physiological and pathological functions of human placenta revealed the hypersecretion of hormones in presence of gestational diseases and raised the question whether this mechanism is cause of disorders of pregnancy, or part of an adaptive response of placenta to resolve adverse conditions. However, there are evidences indicating that changes of placental hormone secretion may have clinical usefulness, since they are measurable in biological fluids, and may be used as predictive markers or prognostic tools. Of particular interest is the role of corticotropin releasing hormone, urocortins and activins in the maintaining physiological pregnancy and in the pathogenesis of diseases (preterm birth and preeclampsia).     

Abundance and nightly activity behavior of a sylvan population of Triatoma dimidiata (Hemiptera: Reduviidae: Triatominae) from the Yucatan,México

Triatoma dimidiata is the vector of Trypanosoma cruzi in the Yucatan Peninsula (YP). Earlier studies have shown that domestic and peri‐domestic populations of the vector originated from the sylvan stock and that effectiveness of insecticide‐spraying was affected by re‐infestations of houses from the sylvan T. dimidiata population. In addition, in the YP most previously published reports have focused on domestic and peri‐domestic populations and very little is known about the nocturnal behavior of the sylvan populations. The main aim of our study was to determine the nightly activity patterns of adult T. dimidiata in a selected location in the YP. Secondly, we sought to document the reproductive status and infection rate of active females. During eight sampling nights spaced from late March to late July, 2007, we collected 544 adult T. dimidiata. We found that square‐cloth illuminated white traps were effective to attract the sylvan individuals and that T. dimidiata adults exhibited a unimodal activity pattern throughout the night. The accumulated mean of captured bugs also showed a non‐linear distribution for females and males. Furthermore, we found that male and female catches were significantly correlated with the means of temperature and humidity recorded during the sampling period. Out of 46 dissected females, we observed that 43.5% of females had fully‐formed eggs in their abdomens, and only two females (4.4%) had sperm within the spermatheca. The infection rate of T. dimidiata harboring T. cruzi was found to be 3.7%. The implications of the light attraction to bugs and potential dispersal capabilities are discussed in the paper in the context of infestation/re‐infestation of rural houses by sylvan T. dimidiata flying adults.