Biomedical subjects

M D'Angelo

Publications and source records attributed to M D'Angelo.

At least 19 recordsLinked to original sources

PubMed2002

Genotyping and antifungal susceptibility of human subgingival Candida albicans isolates.

Subgingival colonization by Candida albicans has been described in human immunodeficiency virus (HIV)-infected individuals, but subgingival isolates have scarcely been characterized, particularly with respect to genotype and antifungal susceptibility. A series of 29 subgingival strains of C. albicans isolated from nine HIV-infected individuals was typed by electrophoretic karyotyping and tested for susceptibility to fluconazole, itraconazole, the new investigational triazole posaconazole and amphotericin B. DNA typing showed genetic heterogeneity within subgingival isolates, as almost every individual harbored his/her own specific isolate. Genetic identity was usually demonstrated within oral and subgingival isolates simultaneously collected from the same individual, but a number of DNA types were found to be unique to subgingival strains. These findings suggest that colonization is not just the result of Candida spreading from oral surfaces, and that subgingivally adapted strains could be involved. All isolates were susceptible to all the triazole drugs tested and amphotericin B. Additional studies on subgingival Candida colonization and further characterization of subgingival isolates are now required to clarify the role of Candida as opportunistic periodontal pathogen.

Adult↗

PubMed2001

Two-photon diffraction and quantum lithography.

We report a proof-of-principle experimental demonstration of quantum lithography. Utilizing the entangled nature of a two-photon state, the experimental results have beaten the classical diffraction limit by a factor of 2. This is a quantum mechanical two-photon phenomenon but not a violation of the uncertainty principle.

Journal Article↗

PubMed2001

p53 displacement from centrosomes and p53-mediated G1 arrest following transient inhibition of the mitotic spindle.

Growing evidence indicates a central role for p53 in mediating cell cycle arrest in response to mitotic spindle defects so as to prevent rereplication in cells in which the mitotic division has failed. Here we report that a transient inhibition of spindle assembly induced by nocodazole, a tubulin-depolymerizing drug, triggers a stable activation of p53, which can transduce a cell cycle inhibitory signal even when the spindle-damaging agent is removed and the spindle is allowed to reassemble. Cells transiently exposed to nocodazole continue to express high levels of p53 and p21 in the cell cycle that follows the transient exposure to nocodazole and become arrested in G(1), regardless of whether they carry a diploid or polyploid genome after mitotic exit. We also show that p53 normally associates with centrosomes in mitotic cells, whereas nocodazole disrupts this association. Together these results suggest that the induction of spindle damage, albeit transient, interferes with the subcellular localization of p53 at specific mitotic locations, which in turn dictates cell cycle arrest in the offspring of such defective mitoses.

Anaphase↗

PubMed2001

Authentic matrix vesicles contain active metalloproteases (MMP). a role for matrix vesicle-associated MMP-13 in activation of transforming growth factor-beta.

Matrix vesicles (MV) play a key role in the initiation of cartilage mineralization. Although many components in these microstructures have been identified, the specific function of each component is still poorly understood. In this study, we show that metalloproteases (MMP), MMP-2, -9, and -13 are associated with MV isolated from growth plate cartilage. In addition, we provide evidence that MV contain transforming growth factor-beta (TGF-beta) and that MV-associated MMP-13 is capable of activating latent TGF-beta. To determine whether MMPs are associated directly with MV, vesicles isolated from growth plate cartilage were sequentially treated with hyaluronidase, NaCl, and bacterial collagenase to remove matrix proteins and other components attached to their outer surface. Finally, the vesicles were incubated with detergent to rupture the MV membrane and expose components that are inside the vesicles. Each treated MV fraction was subjected to substrate zymography, immunoblotting, and substrate activity assay. Whereas active MMP-13 was lost after combined treatment with hyaluronidase and NaCl, MMP-2 and -9 activities were still retained in the pellet fraction even after detergent treatment, suggesting that the gelatinases, MMP-2 and -9, are integral components of MV. In addition, MV contain TGF-beta in the small latent complex, and MMP-13 associated with the MV surface was responsible for activation of TGF-beta. Since the amount of TGF-beta activated by hypertrophic chondrocytes increased with mineral appearance in serum-free chondrocyte cultures, a role for active MV-associated MMPs is suggested in activation of TGF-beta seen during late chondrocyte hypertrophy and mineralization of growth plate cartilage.

Cell Membrane↗

PubMed2001

Effect of antimicrobial mouthrinses on the in vitro adhesion of Candida albicans to human buccal epithelial cells.

Adhesion to epithelial cells is a critical step in successful oral colonization and infection by Candida albicans. Therefore, three mouthrinse products, containing chlorhexidine 0.2% (CHX), cetylpyridinium chloride 0.05% (CPC) or triclosan 0.045% (TRN), were compared for their effects on the in vitro adhesion of C. albicans to human buccal epithelial cells (BEC). Candidal adhesion appeared to be significantly reduced by oral rinsing with the CHX-containing mouthrinse (P<0.0001). In vivo exposure of BEC to the CPC mouthrinse also inhibited adhesion of C. albicans (P<0.0001). Both CHX and CPC products suppressed adhesion to the same extent (P>0.01). On the other hand, the TRN mouthrinse did not significantly affect epithelial adhesion of C. albicans (P>0.01). These findings suggest that mouthrinses containing CHX or CPC could be of value in the control of candidal colonization and infection. Clinical trials are warranted on the effectiveness of these products in reducing oral Candida carriage.

Adult↗

PubMed2001

Characterization of a candidate Trypanosoma brucei U1 small nuclear RNA gene.

We have previously shown that the poly(A) polymerase (PAP) gene of Trypanosoma brucei is interrupted by an intervening sequence. It was postulated that removing this intron by cis-splicing requires a yet unidentified U1 small nuclear RNA (snRNA), which in other organisms engages in base-pair interactions across the 5' splice site during early spliceosome assembly. Here we present a characterization of a 75 nucleotide long candidate T. brucei U1 snRNA. Immunoprecipitation studies indicate that a trimethylguanosine cap structure is present at the 5' end and that the RNA is bound to core proteins common to spliceosomal ribonucleoprotein particles. The U1 snRNA has the potential for extensive intermolecular base pairing with the PAP 5' splice site. We used block replacement mutagenesis to identify sequences necessary for in vivo expression of U1 snRNA. We found that at least two cis-acting elements, tRNA-like A and B boxes, located in the 5'-flanking region are necessary for U1 snRNA synthesis; no internal sequences close to the transcription start site are essential, suggesting a promoter architecture distinct from other trypanosome U-snRNA genes.

5' Untranslated Regions↗

PubMed2001

Reconstitution of cyclin D1-associated kinase activity drives terminally differentiated cells into the cell cycle.

Terminal cell differentiation entails definitive withdrawal from the cell cycle. Although most of the cells of an adult mammal are terminally differentiated, the molecular mechanisms preserving the postmitotic state are insufficiently understood. Terminally differentiated skeletal muscle cells, or myotubes, are a prototypic terminally differentiated system. We previously identified a mid-G(1) block preventing myotubes from progressing beyond this point in the cell cycle. In this work, we set out to define the molecular basis of such a block. It is shown here that overexpression of highly active cyclin E and cdk2 in myotubes induces phosphorylation of pRb but cannot reactivate DNA synthesis, underscoring the tightness of cell cycle control in postmitotic cells. In contrast, forced expression of cyclin D1 and wild-type or dominant-negative cdk4 in myotubes restores physiological levels of cdk4 kinase activity, allowing progression through the cell cycle. Such reactivation occurs in myotubes derived from primary, as well as established, C2C12 myoblasts and is accompanied by impairment of muscle-specific gene expression. Other terminally differentiated systems as diverse as adipocytes and nerve cells are similarly reactivated. Thus, the present results indicate that the suppression of cyclin D1-associated kinase activity is of crucial importance for the maintenance of the postmitotic state in widely divergent terminally differentiated cell types.

Animals↗

PubMed2001

Smad-Runx interactions during chondrocyte maturation.

BACKGROUND: Intracellular signaling triggered by bone morphogenetic proteins (BMPs) results in activated Smad complexes that regulate transcription of BMP-responsive genes. However, the low specificity of Smad binding to regulatory sequences implies that additional tissue-specific transcription factors are also needed. Runx2 (Cbfal) is a transcription factor required for bone formation. We have examined the role of Smads and Runx2 in BMP induction of type X collagen, which is a marker of chondrocyte hypertrophy leading to endochondral bone formation. METHODS: Pre-hypertrophic chondrocytes from the cephalic portion of the chick embryo sternum were placed in culture in the presence or absence of rhBMP-2. Cultures were transiently transfected with DNA containing the BMP-responsive type X collagen promoter upstream of the luciferase gene. The cultures were also transfected with plasmids, causing over-expression of Smads or Runx2, or both. After 24-48 hours, cell extracts were examined for levels of luciferase expression. RESULTS: In the presence of BMP-2, chondrocytes over-expressing BMP-activated Smadl or Smad5 showed significant enhancement of luciferase production compared with that seen with BMP alone. This enhancement was not observed with over-expression of Smad2, a transforming growth factor beta (TGF-beta)-activated Smad. Overexpression of Runx2 in BMP-treated cultures increased transcriptional activity to levels similar to those seen with Smads 1 or 5. When chondrocytes were simultaneously transfected with both Runx2 and Smad 1 or 5, promoter activity was further increased, indicating that BMP-stimulated Smad activity can be augmented by increasing the levels of Runx2. CONCLUSIONS: These results implicate the skeletal tissue transcription factor Runx2 in regulation of chondrocyte hypertrophy and suggest that maximal transcription of the type X collagen gene in pre-hypertrophic chondrocytes involves interaction of BMP-stimulated Smads with Runx2. CLINICAL RELEVANCE: Many skeletal abnormalities are associated with impaired regulation of chondrocyte hypertrophy in growth plates. These studies demonstrate that both BMP-activated Smads and Runx2 levels can modulate chondrocyte transition to hypertrophy.

Animals↗

PubMed2000

Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

Arabidopsis↗

PubMed2000

Gene disruption and basic phenotypic analysis of nine novel yeast genes from chromosome XIV.

In this work, we describe the disruption of nine ORFs of S. cerevisiae (YNL123w, YNL119w, YNL115c, YNL108c, YNL110c, YNL124w, YNL233w, YNL232w and YNL231c) in two genetic backgrounds: FY1679 and CEN.PK2. For the construction of the deletant strains, we used the strategy of short flanking homology (SFH) PCR. The SFH-deletion cassette was made by PCR amplification of the KanMX4 module with primers containing a 5' region of 40 bases homologous to the target yeast gene and with a 3' region of 20 bases homologous to pFA6a-KanMX4 MCS. Sporulation and tetrad analysis of heterozygous deletants revealed that YNL110c, YNL124w and YNL232w are essential genes. The subcellular localization of the protein encoded by the essential gene YNL110c was investigated using the green fluorescent protein (GFP) approach, revealing a nuclear pattern. Basic phenotypic analysis of the non-essential genes revealed that the growth of ynl119w delta haploid cells was severely affected at 37 degrees C in N3 medium, indicating that this gene is required at high temperatures with glycerol as a non-fermentable substrate. The ynl233w delta haploid cells also showed a particular phenotype under light microscopy and were studied in detail in a separate work.

Chromosomes, Fungal↗

PubMed2000

Molecular characterization of canine parvovirus in Brazil by polymerase chain reaction assay.

Canine parvovirus (CPV) was first isolated in 1978 in the USA. Analysis of CPV isolates by monoclonal antibodies and restriction enzymes have shown that after the first emergence of CPV (CPV-2) it evolved to give rise to new antigenic types, which were designated CPV type 2a and type 2b. These new types have replaced the original CPV type 2, although the proportions of each of the new antigenic types vary in different countries. In Brazil, CPV-like infections were first observed in 1979, however, there has been no information concerning the antigenic types of CPV prevailing in South America. In this study, we designed a PCR assay to type canine parvovirus strains in fecal samples collected from symptomatic dogs during 1980 through 1986 and 1990 through 1995. Our data showed that the CPV epizootic in Brazil followed the same pattern observed in the USA of emergence of CPV-2 followed by replacement by the variants CPV-2a and 2b. The predominant strain found during 1980 was CPV-2a, which was substantially replaced by CPV-2b from 1990 to 1995.

Animals↗

PubMed2000

Genomic polymorphism in Uruguayan Creole cattle using RAPD and microsatellite markers.

Uruguayan Creole cattle inhabit areas that cannot sustain conventional farming. They have adapted to fragile environments and are influenced only by natural selection. In this study, random amplified polymorphic DNA (RAPD) and microsatellite (MS) markers were used to analyse Creole cattle genome polymorphism. A comparative analysis using the RAPD technique was performed in pooled DNA of three cattle breeds (Holstein Friesian, Creole and Hereford) in order to evaluate their amplification patterns. A primary screening of RAPD primers allowed us to select and use those with higher percentage of GC base composition. A total of 215 loci ranging between 300 and 2500 bp were amplified. Bandsharing frequency (BSF) among breeds showed that less related fingerprints were observed between Creole and Hereford cattle (0.77), while the highest similarity frequency corresponded to Holstein Friesian compared to Hereford (0.81). Specific RAPD bands were identified in the three DNA pools and they were tested in every individual of each breed. It may be possible to isolate and sequence these bands to create breed-specific molecular markers. The identification of multiple alleles of the MSCYP 21 in Creole cattle with an heterozygosity of He = 0.846 supported the variability of this genetic resource. The use of molecular markers such as RAPD s and microsatellites is proposed to establish genetic distance among American Creole cattle and possibly related ancestral Iberian breeds.

Alleles↗

PubMed2000

Utilization of bone morphogenetic protein receptors during chondrocyte maturation.

Cartilage from the upper, cephalic portion of embryonic chick sternums undergoes hypertrophy, while the lower, caudal portion of the sternum remains as cartilage. Bone morphogenetic proteins (BMPs) induce type X collagen (colX) in cultured upper but not lower sternal chondrocytes (LSCs). We have examined the utilization of BMP receptors (BMPRs) by upper sternal chondrocytes (USCs) and LSCs both by analyzing receptor expression and by overexpressing mutant BMPRs. Reverse-transcription polymerase chain reaction (RT-PCR) analyses indicate that both upper and lower chondrocytes produce messenger RNA (mRNA) for all three receptors: BMPR type IA (BMPR-IA), BMPR type IB (BMPR-IB), and BMPR type II (BMPR-II). Infection of USC with retroviral vectors expressing constitutively active (CA) BMPRs showed that CA-BMPR-IB, like exogenous BMP-4, induced both colX mRNA and elevated alkaline phosphatase (AP), while CA-BMPR-IA was markedly less potent. However, expression of activated receptors in LSC cultures resulted in only minimal induction of hypertrophic markers. Consistent with the results seen for CA receptors, dominant negative (DN) BMPR-IB blocked BMP-induced hypertrophy in USCs more effectively than DN-BMPR-IA. These results imply that the major BMPR required for BMP induction of chondrocyte hypertrophy is BMPR-IB, and that difference between permanent and prehypertrophic chondrocytes is not caused by absence of receptors required for BMP signaling.

Alkaline Phosphatase↗

PubMed2000

MMP-13 is induced during chondrocyte hypertrophy.

During development, mRNA for matrix metalloproteinase-13 (MMP-13) is found associated with cartilage undergoing hypertrophy, suggesting that this collagenase plays a role in cell enlargement and/or cartilage calcification. Using chondrocytes from prehypertrophic cartilage of chick embryo sternae, we have examined the relationship between MMP-13 expression and the transition to hypertrophy. When hypertrophy was induced by serum-free culture with ascorbate and bone morphogenetic protein-2 (BMP-2), MMP-13 mRNA levels paralleled those for type X collagen. Chondrocytes from the caudal, nonhypertrophying portion of chick sternae expressed neither type X collagen nor MMP-13, confirming that MMP-13 mRNA is a marker for hypertrophy. Zymography with conditioned medium yielded a proteinase band at 59 kDa, which was absent in nonhypertrophic chondrocytes. A polyclonal antibody raised against chick MMP-13 reacted with the 59-kDa protein, confirming that it is MMP-13. Although mRNA for MMP-13 peaked at days 4-5 of culture, only low levels of MMP-13 activity were present, and the activity increased gradually in parallel with later increases in MMP-2. These results suggest that MMP-13 is activated by MMP-2 during chondrocyte maturation, and that the combination of both proteinases is required to prepare cartilage matrix for subsequent calcification, before endochondral ossification.

Animals↗

PubMed1999

E2F activates late-G1 events but cannot replace E1A in inducing S phase in terminally differentiated skeletal muscle cells.

We have previously shown that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. Current models imply that much or all of this E1A activity is mediated by the release of the E2F transcription factors from pocket-protein control. In contrast, we show here that overexpression of E2F-1, E2F-2 and E2F-4, or a chimeric E2F-4 tethered to a nuclear localization signal cannot reactivate postmitotic skeletal muscle cells (myotubes). This is not due to lack of transcriptional activity, as demonstrated on both a reporter construct and a number of endogenous target genes. Although cyclin E was strongly overexpressed in E2F-transduced myotubes, it lacked associated kinase activity, possibly explaining the inability of the myotubes to enter S phase and accumulate cyclin A. Although E2F is not sufficient to trigger DNA synthesis in myotubes, its activity is necessary even in the presence of E1A, as dominant-negative DP-1 mutants inhibit E1A-mediated cell cycle reentry. Our data show that, to reactivate myotubes, E1A must exert other functions, in addition to releasing E2F. They also establish mouse myotubes as an experimental system uniquely suited to study the most direct E2F functions in the absence of downstream cell cycle effects.

Adenovirus E1A Proteins↗

PubMed1999

Delayed ischemic preconditioning is mediated by opening of ATP-sensitive potassium channels in the rabbit heart.

Cardioprotection from preconditioning reappears 24 h after the initial stimulus. This phenomenon is called the second window of protection (SWOP). We hypothesized that opening of the ATP-sensitive potassium (KATP) channel mediates the protective effect of SWOP. Rabbits were preconditioned (PC) with four cycles of 5-min regional ischemia each followed by 10 min of reperfusion. Twenty-four hours later, the animals were subjected to sustained ischemia for 30 min followed by 180 min of reperfusion (I/R). Glibenclamide (Glib, 0.3 mg/kg ip) or 5-hydroxydecanoate (5-HD, 5 mg/kg iv) was used to block the KATP channel function. Infarct size was reduced from 41.2 +/- 2. 6% in sham-operated rabbits to 11.6 +/- 1.0% in PC rabbits, a 71% reduction (n = 11, P < 0.01). Treatment with Glib or 5-HD before I/R increased the infarct size to 43.4 +/- 2.6 and 37.8 +/- 1.9%, respectively (P < 0.01 vs. PC group, n = 12/group). Sham animals treated with either Glib or 5-HD had an infarct size of 39.0 +/- 3.4 and 37.8 +/- 1.5%, respectively, which was not different from control (40.0 +/- 3.8%) or sham (41.2 +/- 2.6%) I/R hearts. Monophasic action potential duration (APD) at 50% repolarization significantly shortened by 28.7, 26.6, and 23.3% in sham animals during 10, 20, and 30 min of ischemia. However, no further augmentation in the shortening of APD was observed in PC hearts. Glib and 5-HD significantly suppressed ischemia-induced epicardial APD shortening, suggesting that 5-HD may not be a selective blocker of the mitochondrial KATP channel in vivo. We conclude that SWOP is mediated by a KATP channel-sensitive mechanism that may have occurred because of the opening of the sarcolemmal KATP channel in vivo.

Action Potentials↗

PubMed1999

In situ localization and characterization of active proteases in chronically inflamed and healthy human gingival tissues.

BACKGROUND: Studies have indicated an important role for host-derived proteases in the pathogenesis of periodontal disease. The objectives of this study were: 1) to develop an assay measuring protease activity in situ and 2) to localize and characterize the enzymatic activity in intact inflamed and healthy gingiva. METHODS: Gingival specimens were prepared and over-laid with a quenched fluorescent substrate. Protease activity was visualized by fluorescence microscopy and correlated with histologic features. RESULTS: In inflamed tissues, enzymatic activity was detected mainly in the connective tissue (predominantly matrix metalloproteases) and, to some extent, in the epithelium (predominantly serine proteases). In contrast, clinically healthy tissues failed to exhibit significant amounts of protease activity. Quantitative and qualitative characteristics of protease activity in intact tissues were found to be pH dependent. CONCLUSIONS: The method described here enabled assessment of active proteases in intact tissues where cell-cell and cell-matrix interactions had been maintained. Our results indicate that there are substantial differences in the distribution of specific proteases between clinically healthy and inflamed periodontal tissues.

Adult↗

PubMed1999

[Mid-latency auditory evoked responses in Alzheimer's disease: evaluation of P1 and P3 waves].

A great deal of research has revealed a frequent association between hearing impairment and Alzheimer's disease. Many of these studies have, however, been criticized for the lack of statistical significance, for the methodology used and for doubts regarding the diagnostic criteria used. The lack of uniform results prompted the present research. On the basis of some works in the literature, the authors felt that the study of middle and long-latency auditory evoked potentials, and their expression characterized by the P1 and P3 waves, the appropriate instrument for exploring the cortical and subcortical tracts of the auditory system which are most compromised in Alzheimer's disease. In fact, numerous studies have suggested that wave P1 is generated by peduncle-pontine nucleus cells of the tegmentum and that wave P3 is generated by sites located in the temporal lobes and hippocampus. The present study was conducted on 15 subjects suffering from Alzheimer's disease and 15 controls. Four subjects were excluded from the study because they were affected by Alzheimer's disease with severe dementia and were, thus, unable to cooperate. The 15 controls underwent accurate clinical and instrumental evaluation to rule out any neurological and intellectual disorders. The results for wave P1 show a statistically significant difference between the subjects affected by Alzheimer's disease and the controls. In fact, there was a difference in the presence of this potential. Moreover there was a statistically significant difference in P1 between those patients with average dementia and the controls but not between those with slight dementia and the controls. Finally, comparison of the abnormalities in P1 potential and P3 latency showed that in Alzheimer's disease alterations in P3 arise earlier and are more constant than alterations in P1. The physiopathological meaning of these results is discussed.

Aged↗