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Biomedical subjects

C Mello

Publications and source records attributed to C Mello.

13 recordsLinked to original sources
PubMed2001

Stability of adenoviral vectors following catheter delivery.

Adenoviral vectors have shown promise in a variety of preclinical vascular disease models. Intravascular infusion is one methodology to introduce the adenoviral vector into the affected area of the blood vessel. The biocompatibility of the infusion catheter with the adenoviral vector is key for successful local transfer. It has been recently suggested that catheter-based delivery of adenoviral vectors may result in the loss of vector infectivity. We demonstrate here a catheter capable of delivering adenoviral vectors without the loss of viral particle or infectious titers. First- (DeltaE1) and second- (DeltaE1/DeltaE4) generation adenoviral vectors were tested for their biocompatibility with the Crescendo microporous infusion catheter, which is designed for local infusion of therapeutic agents to human coronary or peripheral arteries. We found that incubation of either the DeltaE1 or the DeltaE1/DeltaE4 viral vectors for up to 30 min in the catheter at 37 degrees C did not result in a loss of viral particles or of viral infectivity. Here, we show that the Crescendo catheter is biocompatible with adenoviral vectors and suitable for vascular gene therapy.

Adenoviridae↗
PubMed2001

A randomized, double-masked, placebo-controlled trial of recombinant granulocyte colony-stimulating factor administration to preterm infants with the clinical diagnosis of early-onset sepsis.

OBJECTIVE: We performed a randomized, double-masked, parallel-groups, placebo-controlled trial of recombinant granulocyte colony-stimulating factor (rG-CSF) administration to 44 preterm neonates who had blood cultures obtained and antibiotics begun because of the clinical diagnosis of early-onset sepsis. Two primary outcome variables were tested 1) mortality and 2) development of nosocomial infections over the 2-week period after dosing. DESIGN AND METHODS: The treatment group (n = 22) received 10 microgram/kg/day of intravenous rG-CSF once daily for 3 days and the placebo group (n = 22) received the same volume of a visually indistinguishable vehicle. Mortality and culture-proven nosocomial infections were recorded. Immediately before the first, second, and third doses, and again 10 days after the first dose, serum concentrations were determined for tumor necrosis factor-alpha, interleukin 6, granulocyte-macrophage colony stimulating factor, and G-CSF, and blood leukocyte counts, absolute neutrophil counts, immature/total neutrophil ratios, platelet counts, and hemoglobin concentrations were measured. RESULTS: The treatment and placebo groups were of similar gestational age (29 +/- 3 vs 31 +/- 3 weeks) and birth weight (1376 +/- 491 vs 1404 +/- 508 g), and had similar Apgar scores and 24-hour Score for Neonatal Acute Physiology scores. The mortality rate was not different between treatment and placebo groups. However, the occurrence of a subsequent nosocomial infection was lower in the rG-CSF recipients (relative risk:.19; 95% confidence interval:.05-.78). rG-CSF treatment did not alter the serum concentrations of the cytokines measured (except for G-CSF). Serum G-CSF levels and blood neutrophil counts were higher in the treatment than in the placebo group 24 hours and 48 hours after dosing. CONCLUSIONS: Administration of 3 daily doses of rG-CSF (10 microgram/kg/day) to premature neonates with the clinical diagnosis of early-onset sepsis did not improve mortality but was associated with acquiring fewer nosocomial infections over the subsequent 2 weeks.

Bacterial Infections↗
PubMed2000

Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference.

In RNA-mediated interference (RNAi), externally provided mixtures of sense and antisense RNA trigger concerted degradation of homologous cellular RNAs. We show that RNAi requires duplex formation between the two trigger strands, that the duplex must include a region of identity between trigger and target RNAs, and that duplexes as short as 26 bp can trigger RNAi. Consistent with in vitro observations, a fraction of input dsRNA is converted in vivo to short segments of approximately 25 nt. Interference assays with modified dsRNAs indicate precise chemical requirements for both bases and backbone of the RNA trigger. Strikingly, certain modifications are well tolerated on the sense, but not the antisense, strand, indicating that the two trigger strands have distinct roles in the interference process.

Animals↗
PubMed1999

Streptavidin-based containment systems for genetically engineered microorganisms.

The use of genetically modified microorganisms for environmental remediation continues to be debated. Conditional lethal systems with tightly regulated gene expression can be used to contain released microorganisms and ameliorate some of the concerns about horizontal gene transfer. We have described streptavidin-based suicide systems to address these concerns and evaluated their function in Pseudomonas putida containing the TOL plasmid for aromatic hydrocarbon metabolism. Tight regulation of expression of a truncated streptavidin gene was required to avoid premature production of the toxic protein. Streptavidin expression was induced by the absence of 3-methyl benzoate (hydrocarbon substrate) which resulted in the elimination of 99.9% of the bacterial culture within eight hours. Low mutant escape rates at 10(-7) per cell per generation were also realized.

Bacteria↗
PubMed1999

Transcriptional repression by the Caenorhabditis elegans germ-line protein PIE-1.

In the early Caenorhabditis elegans embryo, maternally expressed PIE-1 protein is required in germ-line blastomeres to inhibit somatic differentiation, maintain an absence of mRNA transcription, and block phosphorylation of the RNA polymerase II large subunit (Pol II) carboxy-terminal domain (CTD). We have determined that PIE-1 can function as a transcriptional repressor in cell culture assays. By fusing PIE-1 sequences to the yeast GAL4 DNA-binding domain, we have identified a PIE-1 repression domain that appears to inhibit the transcriptional machinery directly. A sequence element that is required for this repressor activity is similar to the Pol II CTD heptapeptide repeat, suggesting that the PIE-1 repression domain might target a protein complex that can bind the CTD. An alteration of this sequence element that blocks repression also impairs the ability of a transgene to rescue a pie-1 mutation, suggesting that this repressor activity may be important for PIE-1 function in vivo.

Amino Acid Sequence↗
PubMed1998

A CBP/p300 homolog specifies multiple differentiation pathways in Caenorhabditis elegans.

Mammalian p300 and CBP are related transcriptional cofactors that possess histone acetyltransferase activity. Inactivation of CBP/p300 is critical for adenovirus E1A to induce oncogenic transformation and to inhibit differentiation, suggesting that these proteins are likely to play a role in cell growth and differentiation. Here we show that a Caenorhabditis elegans gene closely related to CBP/p300, referred to as cbp-1, is required during early embryogenesis to specify several major differentiation pathways. Inhibition of cbp-1 expression causes developmental arrest of C. elegans embryos with no evidence of body morphogenesis but with nearly twice the normal complement of embryonic cells. Mesodermal, endodermal, and hypodermal cells appear to be completely absent in most embryos, however, all of the embryos exhibit evidence of neuronal differentiation. Our analysis of this phenotype suggests a critical role for CBP-1 in promoting all non-neuronal pathways of somatic differentiation in the C. elegans embryo. In contrast, we show that C. elegans genes related to components of a conserved mammalian histone deacetylase, appear to have a role in repressing somatic differentiation. Our findings suggest a model in which CBP-1 may activate transcription and differentiation in C. elegans by directly or indirectly antagonizing a repressive effect of histone deacetylase.

Acetyltransferases↗
PubMed1998

Purification and characterization of recombinant spider silk expressed in Escherichia coli.

A partial cDNA clone, from the 3' end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a 43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk.

Amino Acid Sequence↗
PubMed1997

Initiation of SV40 DNA replication in vitro: analysis of the role played by sequences flanking the core origin on initial synthesis events.

Replication initiation events are suppressed over the SV40 core origin in vitro; they are also greatly reduced over sequences flanking the origin which contain binding sites for several transcription factors. To address the biochemical basis for the gap in initiation events over the flanking sequences, initial synthesis events have been characterized on templates lacking these sequences. Herein, it is demonstrated that previously functional initiation sites are nearly inactive when moved to positions that are proximal to the core origin. Thus, the gap in initiation events depends, in part, on the proximity of the initiation sites to the SV40 core origin. Additional experiments demonstrate that removal of the flanking sequences had little or no effect on DNA unwinding or on the efficiency of initiation of DNA synthesis in vitro. These results indicate that, under our in vitro conditions, initiation of SV40 DNA synthesis is not enhanced by binding of transcription factors to the flanking sequences.

Antigens, Viral, Tumor↗
PubMed1995

Decrements in auditory responses to a repeated conspecific song are long-lasting and require two periods of protein synthesis in the songbird forebrain.

Earlier work showed that playbacks of conspecific song induce expression of the immediate early gene ZENK in the caudo-medial neostriatum (NCM) of awake male zebra finches and that this response disappears with repeated presentations of the same stimulus. In the present study, we investigated whether repetitions of a song stimulus also elicited a decrement in the electrophysiological responses in the NCM neurons of these birds. Multiunit auditory responses in NCM were initially vigorous, but their amplitude decreased (habituated) rapidly to repeated stimulation, declining to about 40% of the initial response during the first 50 iterations. A similar time course of change was seen at the single unit level. This habituation occurred specifically for each song presented but did not occur when pure tones were used as a stimulus. Habituation to conspecific, but not heterospecific, song was retained for 20 h or longer. Injections of inhibitors of protein or RNA synthesis at the recording site did not affect the initial habituation to a novel stimulus, but these drugs blocked the long-term habituation when injected at 0.5-3 h and at 5.5-7 h after the first exposure to the stimulus. Thus, at least two waves of gene induction appear to be necessary for long-lasting habituation to a particular song.

Acoustic Stimulation↗
PubMed1995

Repeated exposure to one song leads to a rapid and persistent decline in an immediate early gene's response to that song in zebra finch telencephalon.

Conspecific song induces a prompt increase in the expression of the zenk gene in the caudo-medial neostriatum (NCM), a part of the auditory telencephalon of songbirds. To test the hypothesis that zenk gene induction in NCM is related to the acquisition of new song-related memories, we presented adult male zebra finches with repeated playbacks of one song. In response, zenk mRNA levels in NCM increased for the first 30 min, but then declined back to baseline levels despite continued stimulation with the same song. When a novel song was then introduced, however, a full zenk response was triggered once again. Even when a full day had passed between the last exposure to a song and the testing period, the song that had been rendered "familiar" by repetition caused no zenk response, whereas a novel song caused a full response. Quantitative analysis indicates that individual cells in NCM must have undergone a selective loss in their zenk responsiveness to the repeated song, while still maintaining their ability to respond to the novel song. These results support the hypothesis that the induction of zenk is related to the formation of long-term memories. Analysis of the stimulus-specific modulation of zenk responses, coupled with appropriate behavioral assays, should provide insight into neural mechanisms responsible for the discrimination and storage of complex perceptual information.

Animal Communication↗
PubMed1985

Caenorhabditis elegans DNA that directs segregation in yeast cells.

We have isolated seven DNA fragments from Caenorhabditis elegans that enhance the mitotic segregation of autonomously replicating plasmids in the yeast Saccharomyces cerevisiae. These segregators, designated SEG1-SEG7, behave like isolated yeast chromosomes: they increase the stability and simultaneously lower the copy number of circular plasmids during mitotic growth in yeast. During meiosis, plasmids containing the C. elegans segregators show higher levels of precocious or aberrant disjunction than do plasmids bearing isolated yeast centromeres. Yet one of the segregators improved the meiotic segregation of the parental plasmid. We estimate that there may be as many as 30 segregator sequences in the C. elegans genome, a value that is consistent with the polycentric nature of C. elegans chromosomes. Five of the seven segregators are linked to sequences that are repeated in the worm genome, and four of these five segregators cross-hybridize. Other members of this family of repetitive DNA do not contain segregator function. Segregator sequences may prove useful for probing the structure of centromeres of both C. elegans and S. cerevisiae chromosomes.

Animals↗