Effects of bisphosphonates on osteogenesis and osteoclastogenesis signaling during the endochondral ossification of growing rats.

Osteoclasts and chondroclasts are necessary, during endochondral ossification, for the resorption of primary bone and calcified cartilage septa, respectively. The bisphosphonates inhibit mineralized tissue resorption by various mechanisms according to the different types of this drug, which can affect bone remodeling during skeletal growth. The objective of the present study is to analyze the way that alendronate (ALN) and etidronate (ETN) can affect osteoclastogenesis and bone formation during endochondral ossification of the long bones of growing rats. Newborn Wistar rats were treated daily with ETN, ALN, or sterile saline solution (control) for 21 days. Their femur and tibiae epiphyses were radiographed and analyzed by light, scanning and transmission electron microscopy. The expression of genes related to osteogenesis and to osteoclast differentiation and activity were analyzed by real-time quantitative polymerase chain reaction. The ETN group presented reduced body weight, disorganized growth plate and an extended area of cartilage in the ossification zone with little bone matrix; in the ALN group, this area was not altered. The ALN presented latent TRAP-positive cells, whereas in the ETN group, they were activated. The expression of NFκB1 and 2, OPG, Spp1 and Runx2 in the ossification zone was reduced by both bisphosphonates. RANKL expression was reduced by ETN, whereas ALN decreased the expression of RANK. The results also indicated that, in addition to the anti-resorptive effect of the drugs, disturbances in bone deposition occurred concomitantly with the reduced expression of osteogenesis-related genes.

The histone genes cluster in Rhynchosciara americana and its transcription profile in salivary glands during larval development.

In this work we report the characterization of the Rhynchosciara americana histone genes cluster nucleotide sequence. It spans 5,131 bp and contains the four core histones and the linker histone H1. Putative control elements were detected. We also determined the copy number of the tandem repeat unit through quantitative PCR, as well as the unequivocal chromosome location of this unique locus in chromosome A band 13. The data were compared with histone clusters from the genus Drosophila, which are the closest known homologues.

Challenges Facing the Use of Endothelial Progenitor Cells in Stem Cell Therapies.

The paper summarizes the difficulties to study the rare population of endothelial progenitor cells in clinical trials, based on the experience of our group in many publications in this area.

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana.

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

The R2 mobile element of Rhynchosciara americana: molecular, cytological and dynamic aspects.

Ribosomal RNA genes are encoded by large units clustered (18S, 5S, and 28S) in the nucleolar organizer region in several organisms. Sometimes additional insertions are present in the coding region for the 28S rDNA. These insertions are specific non-long terminal repeat retrotransposons that have very restricted integration targets within the genome. The retrotransposon present in the genome of Rhynchosciara americana, RaR2, was isolated by the screening of a genomic library. Sequence analysis showed the presence of conserved regions, such as a reverse transcriptase domain and a zinc finger motif in the amino terminal region. The insertion site was highly conserved in R. americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirmed that the RaR2 mobile element was inserted into a specific site in the rDNA gene. The expression level of RaR2 in salivary glands during larval development was determined by quantitative RT-PCR, and the increase of relative expression in the 3P of the fourth instar larval could be related to intense gene activity characteristic of this stage. 5'-Truncated elements were identified in different DNA samples. Additionally, in three other Rhynchosciara species, the R2 element was present as a full-length element.

Molecular characterization of a retrotransposon in the Rhynchosciara americana genome and its association with telomere.

Non-LTR retrotransposons, also known as long interspersed nuclear elements (LINEs), are transposable elements that encode a reverse transcriptase and insert into genomic locations via RNA intermediates. The sequence analysis of a cDNA library constructed from mRNA of the salivary glands of R. americana showed the presence of putative class I elements. The cDNA clone with homology to a reverse transcriptase was the starting point for the present study. Genomic phage was isolated and sequenced and the molecular structure of the element was characterized as being a non-LTR retrotransposable element. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana. Chromosome tips were consistently positive when this element was used as probe in in-situ hybridization. Real-time RT-PCR showed that this retrotransposon is transcribed at different periods of larval development. Most interesting, the silencing of this retrotransposon in R. americana by RNA interference resulted in reduced transcript levels and in accelerated larval development.

Mariner-like elements in Rhynchosciara americana (Sciaridae) genome: molecular and cytological aspects.

Two mariner-like elements, Ramar1 and Ramar2, are described in the genome of Rhynchosciara americana, whose nucleotide consensus sequences were derived from multiple defective copies containing deletions, frame shifts and stop codons. Ramar1 contains several conserved amino acid blocks which were identified, including a specific D,D(34)D signature motif. Ramar2 is a defective mariner-like element, which contains a deletion overlapping in most of the internal region of the transposase ORF while its extremities remain intact. Predicted transposase sequences demonstrated that Ramar1 and Ramar2 phylogenetically present high identity to mariner-like elements of mauritiana subfamily. Southern blot analysis indicated that Ramar1 is widely represented in the genome of Rhynchosciara americana. In situ hybridizations showed Ramar1 localized in several chromosome regions, mainly in pericentromeric heterochromatin and their boundaries, while Ramar2 appeared as a single band in chromosome A.

A shortcut in phage screening technique

A simple modification of the traditional Benton & Davis technique for phage screening is presented that avoids the tedious sample dilutions of putative spots/phages towards the second screening. With the use of a sole agar plate and nylon filter, the modification distinguishes a true positive recombinant from a false positive, with high probability of success.

Molecular characterization of the B-2 DNA puff gene of Rhynchosciara americana.

We have sequenced a 2.5-kb DNA fragment of the B-2 DNA puff from the sciarid Rhynchosciara americana and have defined its transcription unit. This puff is active during the formation of the communal cocoon, which is important for successful metamorphosis of this species and coincides with the final cycle of polytenization in its salivary glands. The B-2 polypeptide, together with the products of two other previously characterized DNA puffs, seems to be engaged in an interaction that results in a gradual modification and hardening of the cocoon structure. The B-2 messenger is temporally regulated in apparent coordination with the other puff products. The predicted polypeptide has characteristics similar to polypeptides from previously sequenced DNA puff genes, in particular those from the R. americana C-8 gene and the Bradysia hygida C-4 gene. The cloned sequence of the B-2 puff is differentially amplified in the three gland regions examined, achieving its highest amplification level of approximately fourfold (two extra cycles) in the anterior segment of the gland. The C-3 DNA puff sequence was also found to be differentially amplified in the different gland regions. Implications of the widespread presence of DNA amplification as a form of gene regulation in the Sciaridae are discussed.

Analysis and functional annotation of an expressed sequence tag collection for tropical crop sugarcane.

To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged.