Interferon Treatment of Ehrlich Ascites Tumor Cells: Effects on Exogenous mRNA Translation and tRNA Inactivation in the Cell Extract

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S30_INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30_INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30_INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30_INT between tRNA from interferon-treated cells and tRNA from control cells. Furthermore, no difference was found in the rate of inactivation in S30_INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under our conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation of exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.     

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

Molecular Biology Reports - Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic...     

Analysis of genome instability in breast cancer

Breast cancer is a heterogeneous disease, previously associated with genomic instability. Our aim was to analyze microsatellite markers in order to determine patterns and levels of instability, as well as possible correlations with histopathological parameters. Polymerase chain reaction was used to characterize microsatellite instability (MSI) and loss of heterozygosity (LOH) in 107 breast carcinomas at twelve microsatellite loci. Some of the markers were selected because of their relation to steroid hormone metabolism, which seems to be related to sporadic breast cancer risk. D5S346 and D17S250 markers showed a statistically significant frequency of MSI. LOH in D3S1611, D17S250, AR and ER-β were associated with some parameters of worse prognosis. Marker group analysis showed that CYP19, AR and ER-β were related to histological grade III, ER-negative and PR-negative cases. Our results suggest that marker group analysis may be preferred to the single marker strategy, being predictive of worst prognosis when single markers are unable to provide such information. A further evaluation of steroid metabolism genes and their association with low penetrance genes in breast cancer may be useful.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Inhibition of vesicular stomatitis virus replication by prostaglandin A1 in Aedes albopictus cells

Cyclopentenone prostaglandins (PGs) exhibit antiviral activity against RNA and DNA viruses in mammalian cell lines, and this effect has been associated with the induction of a heat shock protein (hsp70). We investigated the effect of prostaglandin A1 (PGA1) on the replication of vesicular stomatitis virus (VSV) in Aedes albopictus (mosquito) cells. PGA1 was found to inhibit VSV replication dose dependently. Virus yield was reduced to 50% (3 microg PGA1/ml) and to 95% with 8 microg PGA1/ml. Even with the dramatic reduction of virus production observed in cells treated with PGA1, VSV-specific protein synthesis was unaltered. Treatment of cells with PGA1 (5 microg/ml) stimulated the synthesis of a polypeptide identified as a heat-shock protein (hsp) by immunoblot analysis. PGA1 induced hsp70 synthesis in uninfected cells. However, in VSV-infected cells the induction of hsp70 by PGA1 was reduced. This is the first report of antiviral effects of PGs affecting the replication of VSV in a mosquito cell line.     

Design of bivalent ligands using hydrogen bond linkers: synthesis and evaluation of inhibitors for human beta-tryptase

We exploit the concept of using hydrogen bonds to link multiple ligands for maintaining simultaneous interactions with polyvalent binding sites. This approach is demonstrated by the syntheses and evaluation of pseudo-bivalent ligands as potent inhibitors of human β-tryptase.     

Prospective study of hepatitis C virus infection in hemodialysis patients by monthly analysis of HCV RNA and antibodies

The aims of this study were to (i) evaluate the prevalence and the incidence of hepatitis C virus (HCV) infection in hemodialysis patients in two different centers in S?o Paulo (Brazil), (ii) determine the time required to detect HCV infection among these patients by serology or PCR, (iii) establish the importance of alanine aminotransferase determination as a marker of HCV infection, and (iv) identify the HCV genotypes in this population. Serum samples were collected monthly for 1 year from 281 patients admitted to hospital for hemodialysis. Out of 281 patients, 41 patients (14.6%) were HCV positive; six patients seroconverted during this study (incidence = 3.1/1000 person-month). In 1.8% (5/281) of cases, RNA was detected before the appearance of antibodies (up to 5 months), and in 1.1% (3/281) of cases, RNA was the unique marker of HCV infection. The genotypes found were 1a, 1b, 3a, and 4a. The presence of genotype 4a is noteworthy, since it is a rare genotype in Brazil. These data pointed out the high prevalence and incidence of HCV infection at hemodialysis centers in Brazil and showed that routine PCR is fundamental for improving the detection of HCV carriers among patients undergoing hemodialysis.     

Direct Behavioral and Neurophysiological Evidence for Retronasal Olfaction in Mice

The neuroscience of flavor perception is hence becoming increasingly important to understand food flavor perception that guides food selection, ingestion and appreciation. We recently provided evidence that rats can use the retronasal mode of olfaction, an essential element of human flavor perception. We showed that in rats, like humans, odors can acquire a taste. We and others also defined how the input of the olfactory bulb (OB) -not functionally imageable in humans- codes retronasal smell in anesthetized rat. The powerful awake transgenic mouse, however, would be a valuable additional model in the study of flavor neuroscience. We used a go/no-go behavioral task to test the mouse''s ability to detect and discriminate the retronasal odor amyl acetate. In this paradigm a tasteless aqueous odor solution was licked by water-restricted head-fixed mice from a lick spout. Orthonasal contamination was avoided. The retronasal odor was successfully discriminated by mice against pure distilled water in a concentration-dependent manner. Bulbectomy removed the mice''s ability to discriminate the retronasal odor but not tastants. The OB showed robust optical calcium responses to retronasal odorants in these awake mice. These results suggest that mice, like rats, are capable of smelling retronasally. This direct neuro-behavioral evidence establishes the mouse as a useful additional animal model for flavor research.     

Comprehensive proteomic profiling of adult Angiostrongylus costaricensis, a human parasitic nematode

Angiostrongylus costaricensis is a nematode helminth that causes an intestinal acute inflammatory process known as abdominal angiostrongyliasis, which is a poorly understood human disease occurring in Latin America. Our aim was to study the proteomic profiles of adult parasites focusing on immunogenic proteins. Total cellular extracts from both genders showed similar 2-DE profiles, with 60% of all protein spots focused between pH 5-7 and presenting molecular masses from 20.1 to 66 kDa. A total of 53 different dominant proteins were identified in our dataset and were mainly associated with the following over-represented Gene Ontology Biological Process terms: "macromolecule metabolic process", "developmental process", "response to stress", and "biological regulation". Female and male immunoblots showed similar patterns of reactive proteins. Immunoreactive spots identified by MALDI-PSD were found to represent heat shock proteins, a putative abnormal DAuer Formation family member, and galectins. To date, very few biochemical analyses have focused on the nematode Angiostrongylus costaricensis. As such, our results contribute to a better understanding of its biology and the mechanisms underlying the host-parasite relationship associated with this species. Moreover, our findings represent a first step in the search for candidate proteins for diagnostic assays and the treatment of this parasitic infection.