A proteomic approach to guarana seed and pericarp maturation.

Paullinia cupana Kunth var. sorbilis (Mart.) Ducke, the cultivated guarana plant, is native to the Amazon and has been valued for its medicinal, stimulant and energetic properties for centuries. The seeds are the main commercial product of the plant and the source of high amounts of purine alkaloids (caffeine and theobromine) and polyphenols (flavonoids, catechins, and tannins). Proteins involved in the development and maturation of guarana fruits in its native habitat are interesting issues for proteomics. This study presents the proteomic profile of the seed and pericarp of healthy guarana in different maturation stages. Protein contents were higher in the mature seed compared to other stages due to the accumulation of storage proteins - 11S globulins. Proteins selected for identification by mass spectrometry are mostly related to stress responses and defense and this is not unexpected for fast growing and differentiating reproductive tissues.

Post-transcriptional silencing of the SGE1 gene induced by a dsRNA hairpin in Fusarium oxysporum f. sp cubense, the causal agent of Panama disease.

Fusarium oxysporum f. sp cubense (Foc), the causal agent of Panama disease, is responsible for economic losses in banana crops worldwide. The identification of genes that effectively act on pathogenicity and/or virulence may contribute to the development of different strategies for disease control and the production of resistant plants. The objective of the current study was to analyze the importance of SGE1 gene expression in Foc virulence through post-transcriptional silencing using a double-stranded RNA hairpin. Thirteen transformants were selected based on different morphological characteristics, and sporulation in these transformants was significantly reduced by approximately 95% (P < 0.05) compared to that of the wild-type strain. The relative SGE1 expression levels in the transformant strains were reduced by 27 to 47% compared to those in the wild-type strain. A pathogenicity analysis revealed that the transformants were able to reach the rhizomes and pseudostems of the inoculated banana plants. However, the transformants induced initial disease symptoms in the banana plants approximately 10 days later than that by the wild-type Foc, and initial disease symptoms persisted even at 45 days after inoculation. These results indicate that the SGE1 gene is directly involved in the virulence of Foc. Therefore, SGE1 may be a potential candidate for host-induced gene silencing in banana plants.

Method for obtaining high-resolution proteomic analysis from pericarps of guarana.

Guarana has great agricultural potential and is largely used therapeutically and in the production of non-alcoholic energy drinks. Genomic and proteomic studies are crucial to identify proteins that play central roles in the maintenance and viability of fruits, as well as to identify proteins related to the main metabolic pathways. However, the success of any protein analysis starts with the protein extract preparation, which needs to offer an extract that is free of contaminants. This study aimed to evaluate different extraction methods to obtain high-quantity and high-quality extracts that are compatible with analysis by 2-dimensional electrophoresis and tandem mass spectrometry protein identification. Three different methods were tested: trichloroacetic acid (TCA)/acetone, sodium dodecyl sulfate (SDS)/phenol, and polyvinylpolypyrrolidone (PVPP)/SDS/phenol. The extract obtained from the TCA/acetone precipitation presented low solubility and contamination with lipids and carbohydrates. On the other hand, the quality of the extract gradually improved after using phenol and PVPP/phenol, enabling a yield up to 2 mg/g macerated tissues and the detection of 457 spots by 2-dimensional electrophoresis. The effectiveness of the procedure used was validated by identification of 10 randomly selected proteins by mass spectrometry. The procedure described here can be a starting point for applications using tissues of other organs of guarana or tissues of species that are similar to guarana.

Electrophoresis and spectrometric analyses of adaptation-related proteins in thermally stressed Chromobacterium violaceum.

Chromobacterium violaceum is a Gram-negative proteobacteria found in water and soil; it is widely distributed in tropical and subtropical regions, such as the Amazon rainforest. We examined protein expression changes that occur in C. violaceum at different growth temperatures using electrophoresis and mass spectrometry. The total number of spots detected was 1985; the number ranged from 99 to 380 in each assay. The proteins that were identified spectrometrically were categorized as chaperones, proteins expressed exclusively under heat stress, enzymes involved in the respiratory and fermentation cycles, ribosomal proteins, and proteins related to transport and secretion. Controlling inverted repeat of chaperone expression and inverted repeat DNA binding sequences, as well as regions recognized by sigma factor 32, elements involved in the genetic regulation of the bacterial stress response, were identified in the promoter regions of several of the genes coding proteins, involved in the C. violaceum stress response. We found that 30 °C is the optimal growth temperature for C. violaceum, whereas 25, 35, and 40 °C are stressful temperatures that trigger the expression of chaperones, superoxide dismutase, a probable small heat shock protein, a probable phasing, ferrichrome-iron receptor protein, elongation factor P, and an ornithine carbamoyltransferase catabolite. This information improves our comprehension of the mechanisms involved in stress adaptation by C. violaceum.

Cryopreservation does not alter karyotype, multipotency, or NANOG/SOX2 gene expression of amniotic fluid mesenchymal stem cells.

Cryopreservation of mesenchymal stem cells from amniotic fluid is of clinical importance, as these cells can be harvested during the prenatal period and stored for use in treatments. We examined the behavior of mesenchymal stem cells from human amniotic fluid in culture that had been subjected to cryopreservation. We assessed chromosomal stability through karyotype analysis, determined whether multipotent capacity (differentiation into adipogenic, chondrogenic, and osteogenic cells) is maintained, and analyzed SOX2 and NANOG expression after thawing. Five amniotic fluid samples were cryopreserved for 150 days. No chromosomal aberrations were observed. The expression levels of NANOG and SOX2 also were quite similar before and after cryopreservation. Capacity for differentiation into adipogenic, chondrogenic, and osteogenic tissues also remained the same. We conclude that cryopreservation of amniotic fluid does not alter karyotype, NANOG/SOX2 gene expression, or multipotent capacity of stem cells that have been collected from amniotic fluid during pregnancy.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs.

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger

Stereotactic excisional breast biopsies utilizing the advanced breast biopsy instrumentation system.

OBJECTIVE: To compare the stereotactic excisional breast biopsy ABBI (Advanced Breast Biopsy Instrumentation) system with "open" excisional breast biopsy with needle localization. METHODS: Twenty-three women underwent excisional breast biopsy using the ABBI system, 23 women concomitantly underwent needle localization and excisional breast biopsy. All women had mammograms displaying microcalcifications or nonpalpable noncystic nodular densities suspicious for cancer. RESULTS: Biopsies with ABBI were undertaken with local anesthesia whereas needle localization biopsies were undertaken using general anesthesia. The ABBI system allowed completion mammography. Although preoperative mammograms were comparable, biopsy specimen diameter, volume, and weight were less with ABBI, and patient acceptance was higher. Efficacy, procedural duration, and blood loss were not different between the techniques. CONCLUSIONS: The ABBI system is a minimally invasive yet efficacious excisional breast biopsy technique. It is utilized with local anesthesia in an environment more relaxed and less expensive than the operating room. It allows for smaller biopsy specimens and higher patient acceptance and is as efficacious as needle localization biopsy techniques. The ABBI system belongs in the surgical armamentarium against indeterminant nonpalpable mammographic breast lesions.

Maze performance: a direct comparison of food vs. water mazes.

The purpose of these experiments was to evaluate the utility of a water maze for testing performance in nonfood-restricted rats. Water maze performance was compared to performance in a food-rewarded (food) maze. Separate groups of rats were given single daily trials of 34 days in one of two mazes. The path through each maze was identical; in fact, the same maze was used with the exception that the maze was filled with water during water maze testing and left dry during the food maze testing. In the food maze, a chocolate peanut butter chip was placed at the finish. In the water maze an out-of-the-water platform was placed at the finish. The time to reach the finish was measured for each trial. Both free-feeding and food-restricted rats were tested in each maze. Free-feeding rats learned the food maze with great difficulty, requiring more than 30 trials. Food-restricted rats learned the food maze more quickly than did free-feeding rats. Free-feeding rats learned to solve the water maze more quickly than the food maze. Food-restricted rats also learned the water maze more quickly than the food maze and learned both mazes faster than free-feeding rats. Plasma levels of corticosterone, ACTH and prolactin were measured in all rats immediately following completion of the last maze trial. Plasma corticosterone levels were elevated and plasma prolactin levels were decreased in both food-restricted groups as compared to free-feeding rats, demonstrating that food restriction was chronically stressful.(ABSTRACT TRUNCATED AT 250 WORDS)