A new survey of the serology of human Trypanosoma cruzi infection in the Rio Negro microregion,Brazilian Amazon: a critical analysis

The serology of human Trypanosoma cruzi infection in the Rio Negro microregion is very complex because of the large numbers of false-positive cases that result from low antibody titres and cross-reactions with other infections. In the present study, we collected 4,880 blood samples on filter paper; of these, indirect immunofluorescence (IIF) was strongly reactive in 221 (4.5%), which were considered to be positive (IIF strongly reactive; high intensity of fluorescence) and weakly reactive in 302 (6.2%), which were considered to be doubtful (IIF weakly reactive; low intensity of fluorescence). The confirmatory test on the serum using at least two of three techniques (IIF, conventional ELISA and recombinant ELISA) on 137 samples that were positive in the screening test only confirmed 33 cases (24.1%). Of the 178 samples that were considered doubtful in the screening test, only 10 (5.6%) were considered to be positive in the confirmatory test. Finally, we recommend that the serological diagnosis of T. cruzi infection in the Amazon region be made using at least two different techniques, for example immunofluorescence and ELISA and confirmed by Western blot analysis when possible.     

Imaging the L-Type Amino Acid Transporter-1 (LAT1) with Zr-89 ImmunoPET

The L-type amino acid transporter-1 (LAT1, SLC7A5) is upregulated in a wide range of human cancers, positively correlated with the biological aggressiveness of tumors, and a promising target for both imaging and therapy. Radiolabeled amino acids such as O-(2-[¹⁸F]fluoroethyl)-L-tyrosine (FET) that are transport substrates for system L amino acid transporters including LAT1 have met limited success for oncologic imaging outside of the brain, and thus new strategies are needed for imaging LAT1 in systemic cancers. Here, we describe the development and biological evaluation of a novel zirconium-89 labeled antibody, [⁸⁹Zr]DFO-Ab2, targeting the extracellular domain of LAT1 in a preclinical model of colorectal cancer. This tracer demonstrated specificity for LAT1 in vitro and in vivo with excellent tumor imaging properties in mice with xenograft tumors. PET imaging studies showed high tumor uptake, with optimal tumor-to-non target contrast achieved at 7 days post administration. Biodistribution studies demonstrated tumor uptake of 10.5 ± 1.8 percent injected dose per gram (%ID/g) at 7 days with a tumor to muscle ratio of 13 to 1. In contrast, the peak tumor uptake of the radiolabeled amino acid [¹⁸F]FET was 4.4 ± 0.5 %ID/g at 30 min after injection with a tumor to muscle ratio of 1.4 to 1. Blocking studies with unlabeled anti-LAT1 antibody demonstrated a 55% reduction of [⁸⁹Zr]DFO-Ab2 accumulation in the tumor at 7 days. These results are the first report of direct PET imaging of LAT1 and demonstrate the potential of immunoPET agents for imaging specific amino acid transporters.     

Incidence of toxic Aeromonas isolated from food and human infection

One hundred and ninety four Aeromonas isolates (99 from food and 95 from clinical sources) were analyzed as to the species involved and the toxins produced. Of the clinical isolates of Aeromonas, 29.4% were enterotoxigenic, 43.1% were hemolytic and 89% were cytotoxigenic. Among the food isolates, 18.2% were enterotoxigenic, 17.1% were hemolytic and 72.7% were cytotoxigenic. Aeromonas sobria and Aeromonas veronii produced more enterotoxin and cytotoxin than the other isolates, whereas A. veronii and Aeromonas salmonicida produced cell-free hemolysin. Most of the isolates produced cytotoxins (81%) active on Vero (green monkey kidney) and Chinese hamster ovary cells, but only the culture supernatant of A. sobria produced vacuolation in these cell lines.     

Different signaling pathways in bovine sperm regulate capacitation and hyperactivation

Hyperactivated sperm motility is characterized by high-amplitude and asymmetrical flagellar beating that assists sperm in penetrating the oocyte zona pellucida. Other functional changes in sperm, such as activation of motility and capacitation, involve cross talk between the cAMP/PKA and tyrosine kinase/phosphatase signaling pathways. Our objective was to determine the role of the cAMP/protein kinase A (PKA) signaling pathway in hyperactivation. Western blot analyses of detergent extracts of whole sperm and flagella were performed using antiphosphotyrosine antibody. Bull sperm capacitated by 10 microg/ml heparin and/or 1 mM dibutyryl-cAMP plus 100 microM 3-isobutyl-1-methylxanthine exhibited increased protein tyrosine phosphorylation without becoming hyperactivated. Procaine (5 mM) or caffeine (10 mM) immediately induced hyperactivation in nearly 100% of motile sperm but did not increase protein tyrosine phosphorylation. After 4 h of incubation with caffeine, sperm expressed capacitation-associated protein tyrosine phosphorylation but hyperactivation was significantly reduced. Sperm initially hyperactivated by procaine or caffeine remained hyperactivated for at least 4 h in the presence of Rp-cAMPS (cAMP antagonist) or PKA inhibitors H-89 or H-8. Pretreatment with inhibitors also failed to block induction of hyperactivation; however, the inhibitors did block protein tyrosine phosphorylation when sperm were incubated with capacitating agents, thereby verifying inhibition of the cAMP/PKA pathway. While induction of hyperactivation did not depend on cAMP/PKA, it did require extracellular Ca(2+). These findings indicate that hyperactivation is mediated by a Ca(2+) signaling pathway that is separate or divergent from the pathway associated with acquisition of acrosomal responsiveness and does not involve protein tyrosine phosphorylation downstream of the actions of procaine or caffeine.     

Inhibition of Cytidine Deaminase by Derivatives of 1-(β-D-Ribofuranosyl)-Dihydropyrimidin-2-One (Zebularine)

Abstract

The 2′-deoxy and ara derivatives of 1-β-(D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) were synthesized by improved routes and tested for their inhibitory properties against cytidine deaminase. It was shown that the K_i′s of both compounds were comparable to that of the parent zebularine in inhibition studies with purified enzyme. In contrast to zebularine, 2′-deoxy and ara zebularine showed only nominal cytotoxicity against MOLT-4 and L1210 cells in vitro. A model compound for the inhibition of deoxycytidylate deaminase, 2′-deoxyzebularine 5′-monophosphate (6), was also prepared.     

Conformational Analysis of Nucleosides Constructed on a Bicyclo[3.1.0]hexane Template. Structure-Antiviral Activity Analysis for the Northern and Southern Hemispheres of the Pseudorotational Cycle

Abstract

A conformational analysis of carbocyclic nucleosides built on a rigid bicyclo[3.1.0]hexane template (1–4, Northern and 5–8 Southern) showed that the Northern conformation prefers an anti glycosyl torsion angle whereas the Southern conformation favors the syn range. Antiviral activity was mostly associated with the Northern conformers.     

The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.     

Laser Raman spectra of glucagon

Laser Raman spectroscopy study indicates that in concentrated fresh acidic solution (30 mg/ml), glucagon remains predominantly α-helix and not random-coil. The splitting of the amide III band into three components in the crystal at 1262, 1275, and 1295 cm^?1 is due to the α-conformation as expected. The presence of a small fraction of β-conformation is demonstrated by the appearance of the weak band at 1230 cm^?1 in the fresh solution. This study also established the frequencies of amide III′ bands for the α- and β-conformations of glucagon: 957 and 988 cm^?1 for α and β forms, respectively. The conformations of acidic and basic glucagon solutions are apparently different.