Chromosomal assignment of amplified genes in hydroxyurea-resistant hamster cells

We have shown previously that cDNAs for the M1 and M2 subunits of ribonucleotide reductase, ornithine decarboxylase (ODC), and p5-8, a 55,000-Dalton protein, hybridize to amplified genomic sequences in a highly hydroxyurea-resistant hamster cell line. We have extended these observations to include two additional, independently isolated, hydroxyurea-resistant cell lines: SC8, a single-step hamster ovary cell line, and KH450, a multistep human myeloid leukemic cell line, have also undergone genomic amplification for sequences homologous to ODC and p5-8 cDNAs. However, neither SC8 nor KH450 contains amplified genomic sequences homologous to an M1 cDNA probe. A panel of mouse-hamster somatic cell hybrids was used to map sequences homologous to M1, M2, ODC, and 5-8 cDNAs in the hamster genome. The M2, ODC, and p5-8 cDNAs hybridized to DNA fragments that segregated with hamster chromosome 7. In contrast, M1 cDNA hybridized to DNA fragments that segregated with hamster chromosome 3. These data suggest that the genes RRM2, (M2), ODC, and p5-8, but not RRMI (M1), are linked and may have been co-amplified in the selection of the hydroxyurea-resistant hamster and human cell lines.     

cDNA sequence of the small subunit of the hamster ribonucleotide reductase.

Ribonucleotide reductase activity is markedly elevated in cell lines selected for resistance to hydroxyurea, a cytotoxic drug known specifically to inhibit ribonucleotide reductase. From a cDNA library constructed from a highly hydroxyurea-resistant hamster lung cell line, 600H in which the activity is elevated more than 80-fold, we have isolated a full length cDNA for the small subunit of the reductase. The cDNA is 3.48 kb long with an open reading frame of 1158 nucleotides and a long 3' flanking region of 2169 nucleotides from the termination codon. The derived polypeptide sequence is closely similar to the small subunit of the mouse, differing from it in 20 amino acid positions. Most of these replacements occur in the N-terminal segment of the protein. The hamster subunit does not contain 4 amino acid residues found in the mouse small subunit near the C-terminal end. RNA blots probed with the cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.     

The legacy of forest logging on organic matter inputs and storage in tropical streams

Riparian forests play an important role in stream ecosystems, as they support biodiversity, reduce water erosion, and provide litter that fuels aquatic biota. However, they are affected by great array of anthropogenic threats (e.g., fire, logging, and organic pollution), which alter species composition and their physical structure. Although forest recovery after disturbance such as logging can take decades, the legacy of forest clear-cut logging on key processes in tropical riparian ecosystems is mostly unknown. Here, we investigated how litter inputs (leaves, twigs, and reproductive parts) and storage, key processes for carbon and nutrient recycling and for forest and stream biota, are influenced by riparian vegetation undergoing succession (after 28 years from logging) through the comparison of reference and logged forest sites in the Cerrado biome. Litterfall was overall similar between forest types, but litterfall of twigs was twofold higher at logged than reference sites. Similarly, litter inputs from the bank to the stream (i.e., lateral inputs) and streambed storage were 50–60% higher at logged than reference sites. The higher litterfall observed in logged forests could be related to higher proportion of tree species that are characteristic of primary and secondary successional stages, including fast-growing and liana species, which often are more productive and common in anthropogenic areas. Our results showed that the legacy impact of clear-cut logging, even if residual woody vegetation is maintained in riparian buffers, can shift the type, quantity, and seasonality of litter subsidies to tropical streams. This knowledge should be considered within the context of management and conservation of communities and ecosystem processes in the forest-stream interfaces.     

Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera

BackgroundEnzyme-linked immunosorbent assays (ELISA) are generally the chosen test for Chagas disease (CD) diagnosis; however, its performance depends on the antigen preparation adsorbed to the solid phase, which may lead to false-positive results and cross-reactions. The use of chimeric recombinant antigens can overcome this limitation. Four chimeric antigens from Trypanosoma cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were developed and evaluated in phase I, II and III studies using indirect ELISA as diagnostic platform. However, peroxidase-labeled secondary anti-human IgG antibody, which is employed in indirect ELISAs, limits its use for the detection of species-specific and class-specific antibodies. To overcome this limitation, peroxidase-labeled antigens can be utilized, diagnosing both acute or chronic infection, in a species and immunoglobulin class-independent manner, through the use of a double-antigen sandwich ELISA (DAgS-ELISA). We aimed to evaluate and validate the diagnostic performance of the chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 in the DAgS-ELISA platform.Methodology/Principal findingsDAgS-ELISA was optimized by checkerboard titration. In phase I study, 207 positive and 205 negative samples were evaluated. Cross-reactivity to other infections was also assessed using 68 samples. The selected conditions for the tests utilized 25 ng of antigen per well and the conjugate diluted at 1:2,000 for all molecules. In the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with HTLV.Conclusions/SignificanceDAgS-ELISA is a promising tool for immunodiagnosis, and despite the high AUC values, the performance of this assay was different from the values obtained by our group when using these antigens in the indirect ELISA, for this reason, improvements are being considered to increase the sensitivity of the DAgS-ELISA.     

Impact of Postovulatory Food Deprivation on the Ova Transport, Hormonal Profiles and Metabolic Changes in Sows

The effect of food deprivation on ova transport, hormonal profiles and metabolic changes was studied in 20 crossbred multiparous sows during their second oestrus after weaning. To determine the time of ovulation, transrectal ultrasonographic examination was performed. The sows were divided into 2 groups, one control group (C-group), which was fed according to Swedish standards, and one experimental group (E-group). The E-group sows were deprived of food from the first morning meal after ovulation until slaughter. Blood samples were collected every second hour from about 12 h before expected ovulation in the second oestrus after weaning until slaughter and were analysed for progesterone, prostaglandin F_2α-metabolite, insulin, glucose, free fatty acids and triglycerides. All sows were slaughtered approximately 48 h after ovulation and the genital tract was recovered. The isthmic part of the oviduct was divided into 3 equally long segments and flushed separately with phosphate buffered saline (PBS). Uterine horns were also flushed with PBS. A significantly greater number of ova were found in the first and second part of the isthmus in the E-group (p = 0.05) while in the C-group most of the ova were found in the third part of the isthmus or the uterus (p = 0.01). The level of prostaglandin F_2α-metabolite was significantly higher in the E-group compared with the C-group. The concentration of progesterone increased in both groups after ovulation but there were no significant differences between the groups. The other blood parameters showed that the food-deprived sows were in a catabolic state. The 48 h period of fasting results, directly or indirectly in an delayed ova transport, which may be due to a delayed relaxation in the smooth circular muscle layer of the isthmus.     

Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome

Mitochondrial mutations, an important cause of incurable human neuromuscular diseases, are mostly heteroplasmic: mutated mitochondrial DNA is present in cells simultaneously with wild-type genomes, the pathogenic threshold being generally >70% of mutant mtDNA. We studied whether heteroplasmy level could be decreased by specifically designed oligoribonucleotides, targeted into mitochondria by the pathway delivering RNA molecules in vivo. Using mitochondrially imported RNAs as vectors, we demonstrated that oligoribonucleotides complementary to mutant mtDNA region can specifically reduce the proportion of mtDNA bearing a large deletion associated with the Kearns Sayre Syndrome in cultured transmitochondrial cybrid cells. These findings may be relevant to developing of a new tool for therapy of mtDNA associated diseases.     

Diminazene aceturate associated with sodium selenite and vitamin E in the treatment of Trypanosoma evansi infection in rats

The aim of this study was to evaluate the utilization of a standard treatment with diminazene aceturate against the infection caused by Trypanosoma evansi, associated to sodium selenite and vitamin E. In vitro tests showed trypanocidal effect related to the treatment with diminazene aceturate and sodium selenite, but vitamin E had no harmful effect on the trypanosomes. In vivo experiments utilized a total of 72 adult outbreed females rats, separated into 9 groups (A, B, C, D, E, F, G, H and I), 8 animals each. Group A was the uninfected group; groups B to I were infected with 0.2 mL of blood containing 10⁶ trypanosomes. Parasitemia was estimated daily by microscopic examination of blood smears. Group B served as positive control; group C was treated with diminazene aceturate; group D with sodium selenite; group E with vitamin E; group F received an association of diminazene aceturate and sodium selenite; group G received an association of diminazene aceturate and vitamin E; group H received an association of diminazene aceturate, sodium selenite and vitamin E, and group I received an association of sodium selenite and vitamin E. Diminazene aceturate was administrated in a single dose on the 3rd day post infection (PI). Sodium selenite and vitamin E were administered at the 3rd and 23rd day PI. In vivo tests showed increase of longevity in groups treated with diminazene aceturate associated with sodium selenite (groups F and H). No difference was found between groups C and E, thus the vitamin E did not increase the efficacy of treatment against T. evansi when associated to diminazene aceturate. The curative efficacy of treatments was 37.5, 87.7, 37.7 and 75% to the groups C, F, G and H, respectively. Other treatments showed no efficacy. The sodium selenite when combined with chemotherapy may represent an alternative in the treatment of trypanosomosis.