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L Pereira

Publications and source records attributed to L Pereira.

At least 55 records · Page 3Linked to original sources
PubMed1999

Detection of additional structural variation at the FES/FPS system and population data from S. Tomé e Príncipe and North Portugal.

Comparative analysis of heteroduplex patterns on the STR FES/FPS system led to the detection of a new non-consensus allele 10 in the African population of São Tomé e Príncipe. Automated sequencing confirmed a T-->C substitution at position 177 (1stT of the 6th repeat) which was exclusively found in haplotypic combination with base A in the previously described polymorphic position 34. The new substitution was not detected in a sample from North Portugal. Sequence analysis revealed a triplet of inverted bases, from positions 101 to 103, relative to the sequence described in the GeneBank (Accession No. X06292). This work confirms the capacity of heteroduplex analysis in the detection of DNA structural microvariation and emphasises the complementary utility of manual system analysis and semi-automated techniques for a full characterisation of the genetic variability of STRs.

Adult↗
PubMed1999

Mutations of extracellular matrix components in vascular disease.

BACKGROUND: Marfan's syndrome (MFS) is characterized by manifestations in the skeletal, ocular, and cardiovascular systems. Dilatation of the aortic root is the hallmark feature in the cardiovascular system. Aortic dilatation is associated with fragmented elastic fibers and accumulation of amorphous matrix elements in the medial layer. This pathology is caused by mutations in fibrillin 1, the major structural component of elastic microfibrils. Fibrillin 1 mutations may affect the assembly or function of the elastic microfibrils or both. To answer this important question, MFS-like mice have been created. METHODS: MFS-like mice were generated by homologous gene targeting in embryonic stem cells. Targeting of the mouse fibrillin 1 gene had the dual effect of reducing gene expression 10-fold and of producing an internally deleted protein. RESULTS: Mutant homozygous mice make very small amounts of only mutant fibrillin 1 and die postnatally of MFS-like vascular complications. Histopathological findings include focal fragmentation of elastic fibers and accumulation of amorphous matrix in the aortic media. CONCLUSIONS: A mouse model for the severe form of MFS has been created using the technique of gene targeting. Aside from its clinical value, the model has demonstrated that fibrillin 1 is predominantly involved in the function rather than the assembly of elastic microfibrils.

Animals↗
PubMed1999

The fibrillins.

Fibrillins 1 and 2 are the main constituents of the extracellular microfibrils responsible for the biomechanical properties of most tissues and organs. They are cysteine-rich glycoproteins predominantly made of multiple repeats homologous to the calcium-binding epidermal growth factor module, and are translated as precursor proteins cleaved by furine/PACE-like activities. Fibrillins polymerize extracellularly as parallel bundles of head-to-tail monomers. Binding to calcium rigidifies the structure of the monomers and the supramolecular organization of the macroaggregates. Fibrillin-1 mutations result in the pleiotropic manifestations of Marfan syndrome, and fibrillin-2 alterations cause the overlapping phenotype of congenital contractural arachnodactyly. It is hypothesized that fibrillin-2 guides elastogenesis, whereas fibrillin-1 provides force-bearing structural support. Gene targeting work in the mouse is shedding new light on their distinct and overlapping contributions to tissue morphogenesis and homeostasis. It is also providing an animal model in which to test therapies aimed at reducing hemodynamic stress and the collapse of the aortic matrix during dissecting aneurysm.

Animals↗
PubMed1999

An acidic cluster in the cytosolic domain of human cytomegalovirus glycoprotein B is a signal for endocytosis from the plasma membrane.

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is transported to apical membranes in CMV-infected polarized retinal pigment epithelial (ARPE-19) cells and in Madin-Darby canine kidney (MDCK) epithelial cells constitutively expressing gB. The cytosolic domain of gB contains a cluster of acidic amino acids, a motif that plays a pivotal role in vectorial trafficking in polarized epithelial cells and may also function as a signal for entry into the endocytic pathway. Here we compared gB internalization and recycling to the plasma membrane in CMV-infected human fibroblasts (HF) and ARPE-19 cells by using antibody-internalization experiments. Immunofluorescence and quantitative assays showed that gB was internalized from the cell surface into clathrin-coated transport vesicles and then recycled to the plasma membrane. gB colocalized with clathrin-coated vesicles containing the transferrin receptor in the early endocytic/recycling pathway, indicating that gB traffics in this pathway. The specific role of the acidic cluster in regulating the sorting of gB-containing vesicles in the early endocytic/recycling pathway was examined in MDCK cells expressing mutated gB derivatives. Immunofluorescence assays showed that derivatives lacking the acidic cluster were impaired in internalization and failed to recycle. These findings, together with our earlier observation that the acidic cluster is a key determinant for targeting gB molecules to apical membranes in epithelial cells, establish that this signal is recognized by cellular proteins that participate in polarized sorting and transport in the early endocytic/recycling pathway.

Amino Acid Sequence↗
PubMed1999

Marfan syndrome: new clues to genotype-phenotype correlations.

Fibrillin 1 is the main constituent of extracellular microfibrils. Microfibrils can exist as individual structures or associate with elastin to form elastic fibres. Fibrillin 1 mutations are the cause of the pleiotropic manifestations of the Marfan syndrome (MFS) which principally involve the musculoskeletal, ocular and cardiovascular systems. MFS pathogenesis requires high levels of mutant fibrillin 1 molecules with dominant-negative activity on microfibrillar assembly and function. Gene-targeting experiments in the mouse have shed new light on fibrillin 1 function, genotype-phenotype correlations and aneurysm progression. These experiments have documented the involvement of fibrillin 1 in maintaining tissue homeostasis, suggested the existence of a critical threshold of functional microfibrils for tissue biomechanics, and outlined novel contributors to the pathogenic sequence of vascular wall collapse.

Animals↗
PubMed1999

Geographic heterogeneity of 4 common worldwide cystic fibrosis non-DF508 mutations in Brazil.

Cystic fibrosis (CF) is an autosomal recessive disease caused by at least 750 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of the most common mutation (DF508) in Brazilian patients of European origin is 47%. To determine the frequency of 4 other common CF mutations (G542X, G551D, R553X, and N1303K) in Brazilian patients of European origin, we used direct polymerase chain reaction (PCR) amplification of DNA obtained from dried blood spots on Guthrie cards. The DNA came from 247 non-DF508 chromosomes from 172 Brazilian CF patients ascertained from 5 different states of Brazil. The results show that the 4 mutations account for 17% of the non-DF508 alleles and only 9% of the total number of Brazilian CF alleles. Overall, the frequency of each mutation is different from northern European and North American populations but similar to southern European populations, mainly the Italian and Spanish populations. When Brazilian patients of European origin are grouped according to state of birth, the frequencies of the mutations are significantly different between southern and southeastern states of Brazil. Therefore there are serious implication for risk assessment of DNA-based tests in heterogeneous populations such as Brazilians. Further studies are needed to identify the remaining 44% of CF mutations for the different populations and regions of Brazil.

Adolescent↗
PubMed1998

A novel human cytomegalovirus glycoprotein, gpUS9, which promotes cell-to-cell spread in polarized epithelial cells, colocalizes with the cytoskeletal proteins E-cadherin and F-actin.

Processes by which human herpesviruses penetrate and are released from polarized epithelial cells, which have distinct apical and basolateral membrane domains differing in protein and lipid content, are poorly understood. We recently reported that human cytomegalovirus (CMV) mutants with deletions of the gene US9 formed wild-type plaques in cultures of human fibroblasts but were impaired in the capacity for cell-to-cell spread in polarized human retinal pigment epithelial cells. Unlike the glycoproteins that are required for infection, the protein encoded by CMV US9 plays an accessory role by promoting dissemination of virus across cell-cell junctions of polarized epithelial cells. To identify the product and investigate its specialized functions, we selected Madine-Darby canine kidney II (MDCK) epithelial cells that constitutively express CMV US9 or, as a control, US8. The gene products, designated gpUS9 and gpUS8, were glycosylated proteins of comparable molecular masses but differed considerably in intracellular distribution and solubility. Immunofluorescence laser scanning confocal microscopy indicated that, like gpUS8, gpUS9 was present in the endoplasmic reticulum and Golgi compartments of nonpolarized cells. In polarized epithelial cells, gpUS9 also accumulated along lateral membranes, colocalizing with cadherin and actin, and was insoluble in Triton X-100, a property shared with proteins that associate with the cytoskeleton. We hypothesize that gpUS9 may enhance the dissemination of CMV in infected epithelial tissues by associating with the cytoskeletal matrix.

Actins↗
PubMed1998

Human cytomegalovirus glycoprotein B contains autonomous determinants for vectorial targeting to apical membranes of polarized epithelial cells.

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells grown on permeable filter supports. Laser scanning confocal immunofluorescence microscopy and domain-selective biotinylation of surface membrane domains showed that CMV gB was transported to apical membranes independently of other envelope glycoproteins and that it colocalized with proteins in transport vesicles of the biosynthetic and endocytic pathways. Determinants for trafficking to apical membranes were located by evaluating the targeting of gB derivatives with deletions in the lumen, transmembrane (TM) anchor, and carboxyl terminus. Derivative gB(Delta717-747), with an internal deletion in the luminal juxtamembrane sequence that preserved the N- and O-glycosylation sites, retained vectorial transport to apical membranes. In contrast, derivatives that lacked the TM anchor and cytosolic domain (gBDelta646-906) or the TM anchor alone (gBDelta751-771) underwent considerable basolateral targeting. Likewise, derivatives lacking the entire cytosolic domain (gBDelta772-906) or the last 73 amino acids (gBDelta834-906) showed disrupted apical transport. Site-specific mutations that deleted or altered the cluster of acidic residues with a casein kinase II phosphorylation site at the extreme carboxyl terminus, which can serve as an internalization signal, caused partial missorting of gB to basolateral membranes. Our studies indicate that CMV gB contains autonomous information for apical targeting in luminal, TM anchor, and cytosolic domain sequences, forming distinct structural elements that cooperate in vectorial transport in polarized epithelial cells.

Amino Acid Sequence↗
PubMed1998

Frequencies of the butyrylcholinesterase K mutation in Brazilian populations of European and African origin.

The frequency of the butyrylcholinesterase K mutation was calculated on the basis of data obtained by polymerase chain reaction primer-introduced restriction analysis (PCR-PIRA). The population sample was composed of 177 Brazilians: 95 whites of predominantly European ancestry and 82 admixed individuals (European and African origin). The frequencies--18.4 +/- 2.8% for whites and 17.1 +/- 2.9% for admixed--did not differ from those previously obtained in North America, Scotland, Japan, and Denmark. The occurrence of the K mutation in Europeans, East Asians, and Africans suggests a relatively old origin for this mutation, and the similar frequencies found in these populations may suggest the operation of selective forces.

Black People↗
PubMed1997

Blood lead in Uruguayan children and possible sources of exposure.

Blood samples and questionnaire background data were collected from 96 children (age 2-14 years) living in urban, suburban, or rural areas with varying traffic intensity and industrial lead pollution in Uruguay. Spot samples of tap water were collected from the homes of 44 children, and samples of top soil were taken from seven areas. Samples of air-borne dust were collected in central and suburban Montevideo. Blood lead concentrations (B-Pb) in children ranged between 47 and 191 (mean 96) micrograms/L and exceeded in 36% of the children 100 micrograms/L, the intervention level adopted by the United States Centers for Disease Control. Lead in tap water ranged from 0.2 to 230 (mean 15) micrograms/L and exceeded in 39% of the samples the maximum level recommended by WHO, 10 micrograms/L. Lead pipes were used in parts of the water supply systems. Lead in air varied between different locations from 0.15 to 1.7 micrograms/m3, highest in the very center of Montevideo. The median soil lead ranged from 6 to 2100 micrograms/g and was highest in industrially polluted areas. At multiple regression analysis, B-Pb was significantly associated only with age (P = 0.032) and traffic intensity at school (P = 0.045). No significant impact on B-Pb of lead in water or soil could be established.

Adolescent↗
PubMed1997

Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome.

Aortic aneurysm and dissection account for about 2% of all deaths in industrialized countries; they are also components of several genetic diseases, including Marfan syndrome (MFS). The vascular phenotype of MFS results from mutations in fibrillin-1 (FBN1), the major constituent of extracellular microfibrils. Microfibrils, either associated with or devoid of elastin, give rise to a variety of extracellular networks in elastic and non-elastic tissues. It is believed that microfibrils regulate elastic fibre formation by guiding tropo-elastin deposition during embryogenesis and early post-natal life. Hence, vascular disease in MFS is thought to result when FBN1 mutations preclude elastic fibre maturation by disrupting microfibrillar assembly. Here we report a gene-targetting experiment in mice that indicates that fibrillin-1 microfibrils are predominantly engaged in tissue homeostasis rather than elastic matrix assembly. This finding, in turn, suggests that aortic dilation is due primarily to the failure by the microfibrillar array of the adventitia to sustain physiological haemodynamic stress, and that disruption of the elastic network of the media is a secondary event.

Aortic Dissection↗
PubMed1997

[Value of dacryocystography in localization diagnosis of lacrimal duct stenosis].

PURPOSE: Evaluation of dacryocystography in the preoperative localisation of stenosis of the lacrimal passage. METHOD: The lacrimal system of 20 patients (25 eyes) suffering from lacrimal passage obstruction was examined either by conventional (n = 20) or by digital (n = 5) technique. Diagnostic imaging was evaluated concerning topographic Information of the pathologic lacrimal system, localisation of the level and differential diagnosis of the cause of the obstruction. RESULTS: Cause of the obstruction was chronic dacryocystitis (n = 16), dacryolithiasis (n = 1), atresia of the lacrimal duct (n = 2), posttraumatic lesions (n = 3), rhinosinusitis (n = 1) and carcinoma of the maxillary sinus (n = 1). In 23 of 25 cases (92%) we found an exact correlation between dacryocystography and the intraoperative findings. The variation of stenosis types as well as different examination techniques are presented and compared with literature findings. CONCLUSION: Dacryocystography is a valuable method in the diagnosis and preoperative planing in lacrimal system obstruction.

Adolescent↗
PubMed1997

Endocardial fibroelastosis in a dog.

Endocardial fibroelastosis is an uncommon congenital heart disease in dogs that may be manifested by signs of left-sided congestive heart failure. A three-month-old, male, Fila Brasileiro dog developed signs of generalised heart failure. Physical examination revealed normal temperature, ascites, and pale and cyanotic mucous membranes. The pup died just after radiography which revealed ascites, hepatomegaly, severe cardiac enlargement and pulmonary oedema. At necropsy, serosanguineous fluid in the thorax and abdomen, pulmonary oedema, right ventricular dilatation, hypertrophy and dilatation of the left ventricle, and mitral valve incompetence were observed. The histopathological examination demonstrated that the thickening of the endocardium of the left atrium and left ventricle was due to the presence of elastic and collagen fibres, although there were no signs of an inflammatory process.

Animals↗
PubMed1997

The human cytomegalovirus UL55 (gB) and UL75 (gH) glycoprotein ligands initiate the rapid activation of Sp1 and NF-kappaB during infection.

The cellular transcription factors Sp1 and NF-kappaB were upregulated shortly after the binding of purified live or UV-inactivated human cytomegalovirus (HCMV) to the cell surface. The rapid time frame of transcription factor induction is similar to that seen in other systems in which cellular factors are induced following receptor-ligand engagement. This similarity suggested that a cellular receptor-viral ligand interaction might be involved in Sp1 and NF-kappaB activation during the earliest stages of HCMV infection. To focus on the possible role viral ligands play in initiating cellular events following infection, we first used purified viral membrane extracts to demonstrate that constituents on the membrane are responsible for cellular activation. Additionally, these studies showed, through the use of neutralizing antibodies, that the viral membrane mediators of this activation are the major envelope glycoproteins gB (UL55) and gH (UL75). To confirm these results, neutralizing anti-gB and -gH antibodies were used to block the interactions of these glycoproteins on whole purified virus with their cell surface receptors. In so doing, we found that Sp1 and NF-kappaB induction was inhibited. Lastly, through the use of purified viral gB protein and an anti-idiotypic antibody that mimics the image of the viral gH protein, it was found that the engagement of individual viral ligands with their appropriate cell surface receptors was sufficient to activate cellular Sp1 and NF-kappaB. These results support our hypothesis that HCMV glycoproteins mediate an initial signal transduction pathway which leads to the upregulation of host cell transcription factors and suggests a model wherein the orderly sequence of virus-mediated changes in cellular activation initiates with viral binding via envelope glycoproteins to the cognate cellular receptor(s).

Antibodies, Anti-Idiotypic↗
PubMed1997

Problem-based learning at the University of Southern California School of Dentistry.

Responding to the recent Institute of Medicine report on dental education, the Center for Craniofacial Molecular Biology (CCMB) of the University of Southern California School of Dentistry has developed a parallel track program in dental education leading to the D.D.S. degree. This program was proposed in May of 1995, and the first class of twelve students was admitted in September of that year. Currently two classes are enrolled and plans to admit a further twelve students (Class of 2001) are in place. The educational strategy for this program is totally problem-based. Students work in groups of six with a faculty facilitator, not necessarily a content expert. Facilitators are largely drawn from the multidisciplinary pool of research faculty at the center. All learning is mediated through biomedical and biodental problem cases. No formal lectures or classes are scheduled. The learning of clinical dental skills is promoted through focussed dental patient simulations in which students review clinical charts, radiographs, medical reports and then explore identified, hands-on learning needs using patient simulators in a clinical context. Early patient exposure is obtained through dental office visits and other special patient clinics. Initial experience with this program suggests that the problem-based learning (PBL) students learn as well (if not better) than their traditional program peers and develop excellent group and cognitive analytical skills. The absence of a pool of dentally related biomedical cases suitable for a PBL program has necessitated the use of innovative approaches to their development and presentation. It is believed that this educational approach will produce dental clinicians equipped with the self-motivated, life-long learning skills required in the ever-changing world of bio-dental sciences in the twenty-first century.

California↗
PubMed1997

Humoral immune response to functional regions of human cytomegalovirus glycoprotein B.

Sera from patients with primary human cytomegalovirus (HCMV) infections, both acute and convalescent phase, and from HCMV-seropositive healthy subjects were analyzed to determine whether the sera would recognize antigenic domains on HCMV glycoprotein B (gB) that function in virion infectivity and spread of virus from cell to cell. The intact gB molecule, amino-terminal derivatives of different lengths, and internal deletion derivatives were expressed in eukaryotic cells and reacted by immunofluorescence with the sera. All convalescent-phase sera and most sera from healthy seropositive individuals reacted with full-length gB and with an amino-terminal derivative containing 687 amino acids (aa), gB-(1-687); approximately half of the sera recognized an amino-terminal derivative of 447 aa, gB-(1-447), and one-third reacted with the shortest deletion derivative of 258 aa, gB-(1-258). Of the acute-phase sera, 77% recognized intact gB and gB-(1-687), 32% recognized gB-(1-447), and 14% recognized gB-(1-258). Deletion of aa 548 to 618 dramatically reduced the percentage of reactive sera, whereas deletion of aa 411 to 447 had a minor impact on reactivity of sera. To investigate the epitope specificity of human antibodies to gB, we carried out competition experiments between human sera with neutralizing activity and selected monoclonal antibodies (mAbs) to conformational epitopes on gB. We found that antibodies in human sera preclude syncytium formation in UB15-11 glioblastoma cells constitutively expressing gB and compete with certain murine mAbs that block virus entry into cells and transmission of infection from cell to cell. Our results show that HCMV-immune human sera contain antibodies to functional regions on gB, and the abundance of these antibodies in convalescent-phase sera suggests that they may play a central role in limiting dissemination of virus in the host.

Acute Disease↗
PubMed1996

Failed PCR amplifications of MBP-STR alleles due to polymorphism in the primer annealing region.

The PCR-based STR system MBP-B (myelin basic protein locus B) has been reported to exhibit a high rate of mutations. Using a newly designed pair of primers we present evidence that this is due to failed amplifications caused by a polymorphism in the annealing region of the reverse primer originally designed. With the new reverse primer described here no exclusions were found (out of 59 mother/child pairs analysed) while one was detected with the old set of primers. The results obtained with both pairs of primers in a random population sample (n = 112) from North Portugal are compared. In this sample 13 individuals typed as homozygotes with the pair of primers originally described, were found to be heterozygous when the amplifications were performed with the new reverse primer. By sequence analysis, a substitution in the reverse primer binding sequence originally described was determined. This substitution is located upstream from the repetition site and consists of G-->A transition. This variation reaches polymorphic frequency and is responsible for the relatively frequent null alleles due to failed amplifications when the previously designed primers are used.

Alleles↗