Search PubMed⌕ Search

Biomedical subjects

D Stern

Publications and source records attributed to D Stern.

At least 37 records · Page 2Linked to original sources
PubMed1999

The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.

A fundamental goal of genetics and functional genomics is to identify and mutate every gene in model organisms such as Drosophila melanogaster. The Berkeley Drosophila Genome Project (BDGP) gene disruption project generates single P-element insertion strains that each mutate unique genomic open reading frames. Such strains strongly facilitate further genetic and molecular studies of the disrupted loci, but it has remained unclear if P elements can be used to mutate all Drosophila genes. We now report that the primary collection has grown to contain 1045 strains that disrupt more than 25% of the estimated 3600 Drosophila genes that are essential for adult viability. Of these P insertions, 67% have been verified by genetic tests to cause the associated recessive mutant phenotypes, and the validity of most of the remaining lines is predicted on statistical grounds. Sequences flanking >920 insertions have been determined to exactly position them in the genome and to identify 376 potentially affected transcripts from collections of EST sequences. Strains in the BDGP collection are available from the Bloomington Stock Center and have already assisted the research community in characterizing >250 Drosophila genes. The likely identity of 131 additional genes in the collection is reported here. Our results show that Drosophila genes have a wide range of sensitivity to inactivation by P elements, and provide a rationale for greatly expanding the BDGP primary collection based entirely on insertion site sequencing. We predict that this approach can bring >85% of all Drosophila open reading frames under experimental control.

Alleles↗
PubMed1999

EMAP II: a modulator of neovascularization in the developing lung.

Neovascularization is a key regulatory process in fetal growth and development. Although factors promoting growth and development of the pulmonary vasculature have been investigated, nothing is known regarding the molecular mechanisms that may counteract these stimuli. Endothelial monocyte-activating polypeptide (EMAP) II has recently been identified as an antiangiogenic factor in tumor vascular development. We postulated that EMAP II is a putative negative modulator of lung vascular growth. EMAP II mRNA and protein decrease fivefold (P < 0.01) as the developing lungs in the fetal mouse progress from having poor vascularization (day 14) to having complete vascular development at term (day 18.5). EMAP II protein expression continues to remain low throughout postnatal life and into adulthood, with the exception of a surge that correlates with microvascular maturation. Furthermore, through the use of in situ hybridization and immunohistochemistry, EMAP II is localized throughout the lung, with significant expression in the submyoepithelial area during the early stages of lung development when there is minimal vascular development. In contrast, EMAP II is distributed around the large vessels during the end of vascular development, suggesting that EMAP II modulates the neovascularization process. We speculate that EMAP II is a director of neovascularization in the developing lung.

Animals↗
PubMed1998

Two color hybridization analysis using high density oligonucleotide arrays and energy transfer dyes.

High density oligonucleotide arrays (DNA chips) have been used in two color mutational analysis of the 3.43 kb exon 11 of the hereditary breast and ovarian cancer gene BRCA1 . Two color analysis allows competitive hybridization between a reference standard and an unknown sample, improving the performance of the assay. Fluorescein and phycoerythrin dyes werepreviously used due to their compatibility with a single line 488 nm excitation source. Here we show that an alternative dye combination, containing the energy transfer dye system phycoerythrin*cy5 along with phycoerythrin, provides more evenly matched signal intensities and decreased spectral overlap between the two fluorophores, while maintaining compatibility with a 488 nm excitation source.

Base Sequence↗
PubMed1998

Human blood-brain barrier receptors for Alzheimer's amyloid-beta 1- 40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer.

A soluble monomeric form of Alzheimer's amyloid-beta (1-40) peptide (sAbeta1-40) is present in the circulation and could contribute to neurotoxicity if it crosses the brain capillary endothelium, which comprises the blood-brain barrier (BBB) in vivo. This study characterizes endothelial binding and transcytosis of a synthetic peptide homologous to human sAbeta1-40 using an in vitro model of human BBB. 125I-sAbeta1-40 binding to the brain microvascular endothelial cell monolayer was time dependent, polarized to the apical side, and saturable with high- and low-affinity dissociation constants of 7.8+/-1.2 and 52.8+/-6.2 nM, respectively. Binding of 125I-sAbeta1-40 was inhibited by anti-RAGE (receptor for advanced glycation end products) antibody (63%) and by acetylated low density lipoproteins (33%). Consistent with these data, transfected cultured cells overexpressing RAGE or macrophage scavenger receptor (SR), type A, displayed binding and internalization of 125I-sAbeta1-40. The internalized peptide remains intact > 94%. Transcytosis of 125I-sAbeta1-40 was time and temperature dependent, asymmetrical from the apical to basolateral side, saturable with a Michaelis constant of 45+/-9 nM, and partially sensitive to RAGE blockade (36%) but not to SR blockade. We conclude that RAGE and SR mediate binding of sAbeta1-40 at the apical side of human BBB, and that RAGE is also involved in sAbeta1-40 transcytosis.

Alzheimer Disease↗
PubMed1998

Tissue factor transcription driven by Egr-1 is a critical mechanism of murine pulmonary fibrin deposition in hypoxia.

Local hypoxemia and stasis trigger thrombosis. We have demonstrated previously that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a result of expression of tissue factor, especially in oxygen-deprived mononuclear phagocytes (MPs). We now show that transcription factor early-growth-response gene product (Egr-1) is rapidly activated in hypoxia, both in vitro and in vivo, and is responsible for transcription and expression of tissue factor in hypoxic lung. MPs and HeLa cells subjected to hypoxia (pO2 approximately 13 torr) had increased levels of tissue factor transcripts (approximately 18-fold) and an increased rate of transcription (approximately 15-fold), based on nuclear run-on analysis. Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells demonstrated increased DNA-binding activity at the serum response region (SRR; -111/+14 bp) of the tissue factor promoter at Egr-1 motifs. Using 32P-labeled Egr consensus oligonucleotide, we observed induction of DNA-binding activity in nuclear extracts from hypoxic lung and HeLa cells because of activation of Egr-1, by means of supershift analysis. Transient transfection of HeLa cells with chimeric plasmids containing wild-type or mutant SRR from the tissue factor promoter showed that intact Sp1 sites are necessary for basal promoter activity, whereas the integrity of Egr-1 sites was required for hypoxia-enhanced expression. A central role for Egr-1 in hypoxia-mediated tissue factor expression was confirmed by experiments with homozygous Egr-1 null mice; wild-type mice subjected to oxygen deprivation expressed tissue factor and showed fibrin deposition, but hypoxic homozygous Egr-1 null mice displayed neither tissue factor nor fibrin. These data delineate a novel biology for hypoxia-induced fibrin deposition, in which oxygen deprivation-induced activation of Egr-1, resulting in expression of tissue factor, has an unexpected and central role.

Animals↗
PubMed1998

Suppression of accelerated diabetic atherosclerosis by the soluble receptor for advanced glycation endproducts.

Accelerated atherosclerosis in patients with diabetes is a major cause of their morbidity and mortality, and it is unresponsive to therapy aimed at restoring relative euglycemia. In hyperglycemia, nonenzymatic glycation and oxidation of proteins and lipids results in the accumulation of irreversibly formed advanced glycation endproducts. These advanced glycation endproducts engage their receptor in cells of the blood vessel wall, thereby activating mechanisms linked to the development of vascular lesions. We report here a model of accelerated and advanced atherosclerosis in diabetic mice deficient for apolipoprotein E. Treatment of these mice with the soluble extracellular domain of the receptor for advanced glycation endproducts completely suppressed diabetic atherosclerosis in a glycemia- and lipid-independent manner. These findings indicate interaction between the advanced glycation endproducts and their receptor is involved in the development of accelerated atherosclerosis in diabetes, and identify this receptor as a new therapeutic target in diabetic macrovascular disease.

Animals↗
PubMed1998

Clinical-radiological evaluation of poststernotomy wound infection.

The presumption that computed tomography is the "gold standard" imaging method for diagnosing poststernotomy sternal wound infection was never validated. This study was designed to evaluate the accuracy and role of computed tomography in diagnosing the extent of infectious complications following sternotomy. A high postoperative infection recurrence rate in our earliest cases (30 percent, 1984 to 1988) motivated us to assess whether this modality enables the surgeon to choose the optimal surgical approach, which will make it possible to reduce morbidity and mortality rates. Two-hundred three patients with poststernotomy sternal wound infections were operated upon between 1984 and 1993. All pertinent clinical and radiological data of these patients were collected retrospectively and reinterpreted by an unbiased radiologist; the radiological data were correlated both to the intraoperative clinical findings and to histological interpretation of the surgical specimens. The study group available for statistical analysis included 160 patients. Predictive statistical analysis confirmed that computed tomography is a highly reliable imaging method for identifying the different pathologies as soft tissue, sternum mediastinal infections, in sternal wound infection with overall sensitivity of 93.5 percent and specificity of 81.7 percent. New radiographic findings were identified for the distinction of costochondral infection. This complication was, and still is, a major deceptive clinical problem in these patients and the major contributor to recurrences. We propose a sternal wound infection classification system that outlines the recommended approach for each clinical-radiological condition. Since computerized tomography was found to be a highly accurate modality, we strongly believe that the surgeon should take its pathological-radiographic findings into serious consideration, even if there are no "clear-cut" clinical signs for an existing or recurring infection.

Adolescent↗
PubMed1998

Risk factors associated with human cystic echinococcosis in Florida, Uruguay: results of a mass screening study using ultrasound and serology.

Sonographic evidence of asymptomatic Echinococcus granulosus lesions in the liver was found in 156 of 9,515 persons in the Department of Florida, Uruguay. The sensitivity of ELISA and latex agglutination serology compared with ultrasound was 47.6% and 28.1%, respectively, and specificity was > 85%. There was a significant positive association between positive sonography and a personal history of previous but treated Echinococcus infection while those that were seropositive but ultrasound-negative were significantly more likely to have a personal history of infection or a history of infection in their family. Prevalence of infection increased significantly with age. There was no correlation between echinococcosis and dog ownership or home slaughter of sheep but offal disposal was important, with an increased prevalence of infection of 3.2%, 2.8%, and 3.1%, respectively, in persons feeding offal to dogs or burying or burning it compared with a prevalence of 0.8-1.5% in those using other methods of disposal. Almost half the population, when questioned, seemed to have sound knowledge about E. granulosus and described correct treatment of E. granulosus in dogs but this did not affect prevalence. There was a significant positive association between infection and the presence of a fenced fruit/vegetable garden and use of rural waters, particularly the cachimba (a small dam) and the aljibe (a cistern or tank) that collect rainwater from the ground surface and roofs, respectively.

Adolescent↗
PubMed1998

Mothers' emotional needs.

In most families, mothers contribute substantially to the new infant's emotional environment and development. Because such mother-infant interaction is crucial, a mother's emotional context is very relevant to infant development. This article in New Perspectives in Early Emotional Development addresses the relationships that a mother requires to regulate her maternal or parental capacities, which enable the infant to develop appropriately.

Emotions↗
PubMed1997

An intracellular protein that binds amyloid-beta peptide and mediates neurotoxicity in Alzheimer's disease.

Amyloid-beta is a neurotoxic peptide which is implicated in the pathogenesis of Alzheimer's disease. It binds an intracellular polypeptide known as ERAB, thought to be a hydroxysteroid dehydrogenase enzyme, which is expressed in normal tissues, but is overexpressed in neurons affected in Alzheimer's disease. ERAB immunoprecipitates with amyloid-beta, and when cell cultures are exposed to amyloid-beta, ERAB inside the cell is rapidly redistributed to the plasma membrane. The toxic effect of amyloid-beta on these cells is prevented by blocking ERAB and is enhanced by overexpression of ERAB. By interacting with intracellular amyloid-beta, ERAB may therefore contribute to the neuronal dysfunction associated with Alzheimer's disease.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1997

Amyloid-beta peptide-receptor for advanced glycation endproduct interaction elicits neuronal expression of macrophage-colony stimulating factor: a proinflammatory pathway in Alzheimer disease.

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloid-beta peptide (Abeta) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Abeta, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor kappaB-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Abeta deposits, and in cerebrospinal fluid from AD patients there was approximately 5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by Abeta-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to Abeta, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Abeta on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD.

Alzheimer Disease↗
PubMed1997

Replacement of Pro109 by His in TlpA, a thioredoxin-like protein from Bradyrhizobium japonicum, alters its redox properties but not its in vivo functions.

TlpA, the membrane-anchored, thioredoxin-like protein from Bradyrhizobium japonicum, is essential for cytochrome aa3 biogenesis. The periplasmic domain of TlpA was previously shown to have protein thiol:disulfide oxidoreductase activity and reducing properties similar to those of cytoplasmic thioredoxins. Here, we replaced the proline-109 in its active-site sequence C107 V108 P109 C110 by a histidine residue. The resulting active-site motif (CVHC) resembles that of oxidizing thiol:disulfide oxidoreductases such as protein disulfide isomerase (PDI) and DsbA. Indeed, the TlpA variant P109H was by 66 mV more oxidizing than the wild-type protein. Nevertheless, the altered protein was even more efficient in catalyzing the reduction of insulin disulfides by dithiothreitol than the wild-type due to a faster recycling of its catalytically active, reduced form. Cells of B. japonicum expressing only the mutated tlpA gene had the same phenotypes as wild-type cells, suggesting that the change in the redox potential of TlpA was not critical for its in vivo function.

Amino Acid Sequence↗
PubMed1997

Principles of the surgical approach in human liver cystic echinococcosis.

Ultrasonography (USG) has recently contributed much to the diagnostic of hepatic cystic echinococcosis (CE). The use of portable ultrasonograph allowed us to perform a community survey among 9482 people living in a high risk area for CE in the Florida Department (Uruguay). Positive USG results were found in 123 asymptomatic patients. 48 out of 51 USG positive cases were surgically confirmed and 3 were found to be false positive. The results of this survey allowed us to propose a new classification of the echographic imaging based on the parasite's various evolutive and involutive stages. The Echinococcus granulosus cyst size was compared with the parasite's evolutive stages. The cyst's segmentary topography and the related risk of CE is evaluated. The importance of cystic-biliary communication is pointed out and its rational surgical treatment described. Finally, an algorithm is presented facilitating the choice of a rational treatment.

Adolescent↗
PubMed1997

Nuclear factor interleukin 6 motifs mediate tissue-specific gene transcription in hypoxia.

Activation of transcription at the nuclear factor interleukin 6 (NF-IL-6) DNA binding motif modulates expression of multiple genes important in host adaptive and developmental mechanisms. Studies showing that hypoxia-induced transcription of IL-6 in cultured endothelial cells was due to transcriptional activation by the NF-IL-6 motif in the promoter (Yan, S.-F., Tritto, I., Pinsky, D., Liao, H., Huang, J., Fuller, G., Brett, J., May, L., and Stern, D. (1995) J. Biol. Chem. 270, 11463-11471) led us to prepare transgenic mice using 115- or 14-base pair regions of the promoter encompassing the NF-IL-6 site ligated to the lacZ reporter gene and the basal thymidine kinase promoter. On exposure to hypoxia or induction of ischemia, mice bearing either of the constructs showed prominent expression of the transgene in lung and cardiac vasculature and in the kidney but not in the liver (parenchyma or vasculature). In contrast, transgenic mice bearing a mutationally inactivated NF-IL-6 site showed no increase in transgene expression in hypoxia. Gel retardation assays revealed time-dependent, hypoxia-enhanced nuclear binding activity for the NF-IL-6 site in nuclear extracts of the heart, lung, and kidney but not in the liver; the hypoxia-enhanced band disappeared on addition of antibody to C/EBPbeta-NF-IL-6. Consistent with the specificity of hypoxia-mediated activation of C/EBPbeta-NF-IL-6, gel retardation assays showed no change in the intensity of the hypoxia-enhanced gel shift band in the presence of excess unlabeled oligonucleotide probes or antibodies related to other transcription factors, including NFkappaB, AP1, cAMP response element-binding protein, SP1, and hypoxia-inducible factor 1. These data indicate that the transcription factor NF-IL-6 is sensitive to environmental oxygen deprivation, and the tissue-specific pattern of gene expression suggests that local mechanisms have an important regulatory effect.

Animals↗
PubMed1997

Capillary and aortic endothelia interact in situ with nonenzymatically glycated albumin and develop specific alterations in early experimental diabetes.

Diabetic mice (6 weeks duration) were studied to assess the interaction of advanced glycation endproduct-modified albumin (AGE-Alb) with micro- and macrovascular endothelium, and to evaluate the alterations induced in the ultrastructure of the lung, kidney, and aorta. [125I]-AGE-Alb and AGE-Alb-Au were perfused in situ in the vasculature; the total uptake was quantitated by spectrometry, and the endothelial pathways of AGE-Alb-Au and the morphological alterations of the vascular beds were examined by electron microscopy. The results showed that [125I]-AGE-Alb (0.567 microM) was taken up specifically and saturably by all organs studied, and particularly by the lung. AGE-Alb-Au endocytosis and transcytosis occurred in the pulmonary and aortic endothelia, and were enhanced in diabetic animals. Also in diabetic animals, AGE-Alb-Au was detected throughout the kidney glomerular basement membrane (GBM) and within open filtration slits of podocytes, suggesting altered barrier function. The structural modifications progressed, and at the end of the experimental period, in the lung approximately 28% of the capillaries and approximately 25% of the alveoli became compressed or even collapsed, due to the hyperplasia of extracellular matrix and interstitial connective tissue. The presence of adherent intravascular macrophages suggests the development of an inflammatory immune process. The structural modifications observed in kidney glomeruli included thickening (approximately 30%) of the GBM and the disappearance of diaphragms between the cellular processes of podocytes. The aortic endothelium displayed luminal foldings, increased number (2.8-fold) of Weibel-Palade bodies, and proliferation of basal lamina. Together, the results show that in diabetes there is enhanced vascular uptake of AGE-Alb and significant pathomorphological changes of micro- and macrovessels.

Animals↗
PubMed1997

Activation of naive xenogeneic but not allogeneic endothelial cells by human naive neutrophils: a potential occult barrier to xenotransplantation.

Here we demonstrate that human neutrophils, the predominant circulating leukocytes in intimate contact with endothelial cells lining the vasculature, directly recognize xenogeneic endothelium independently of xenoreactive natural antibody and complement. A rapid and calcium-dependent activation of native (unstimulated) xenogenic endothelial cells by human neutrophils leads to 1) translocation of P-selectin from the Wiebel-Palade bodies to the surface of xenogeneic endothelial cells, 2) increased synthesis and expression of vascular cell adhesion molecule-1 on the xenogeneic endothelial cells, and 3) enhanced killing of the xenogeneic endothelium by natural killer cells. Our data directly implicate naive neutrophils as major early participants in xenograft recognition and endothelial activation independent of xenoreactive natural antibodies and complement.

Animals↗
PubMed1997

Pathobiology of the heart in experimental diabetes: immunolocalization of lipoproteins, immunoglobulin G, and advanced glycation endproducts proteins in diabetic and/or hyperlipidemic hamster.

Diabetes is known to be accompanied by atherosclerotic disease and general cardiovascular complications. Hamsters were previously shown to develop hyperlipemia-induced atherosclerosis, similar in many respects to the human atherosclerotic process. To study the effect of hyperglycemia on heart vessels and valves, male Golden Syrian hamsters were rendered either diabetic or hyperlipemic and diabetic; controls were age-matched normal hamsters. At time intervals ranging from 2 to 24 weeks, animals were killed; plasma glucose, cholesterol, and lipid peroxides were measured; and the aortic arch and valves, coronary arteries, and heart microvessels were examined for ultrastructural modifications and for the presence of low-density lipoproteins (LDL), immunoglobulin G (IgG), and advanced glycation endproducts (AGE) proteins. Elevation of plasma glucose, peroxides, and cholesterol were observed in both diabetic as well as hyperlipemic and diabetic animals, along with characteristic diabetic changes: microangiopathy of the myocardium (ie, capillary narrowing, hyperplasia of basal lamina, and proliferation of extracellular matrix) and macroangiopathy of the aortic arch, valves, and coronary arteries (ie, intimal proliferation, fatty-streak formation, and calcification). LDL, IgG, and AGE-proteins were immunolocalized in focal deposits, ie, in the shoulder and cap of the plaques; these antigens were distributed diffusely in the extracellular space or within macrophage-derived foam cells and smooth muscle cells. Our findings indicate that hyperglycemia alone induces atherosclerotic lesions in the coronary arteries, aortic arch, and aortic valves as well as alterations of the extracellular matrix of heart microvessels and cardiomyocytes, changes which together may lead to cardiomyopathy, a common and severe complication of diabetes. In addition, the present study suggests that when hyperglycemia is accompanied by hyperlipemia, detectable amounts of modified LDL (possibly oxidized or glycated) and AGE are present in the intima of atherosclerotic arteries; and also that modified lipoproteins can act as immunoactive components of the atheroscerotic process generated by hyperglycemia.

Animals↗