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At least 181 records · Page 10Linked to original sources
PubMed1994

Detection of blood chimerism after lung and heart-lung transplantation. The superiority of nested as compared with standard polymerase chain reaction amplification.

Migration of donor cells from the graft to various tissues of the recipient has been demonstrated after different types of solid organ transplants. Currently, the detection of donor cells in the recipient's tissues is most simply performed by polymerase chain reaction (PCR) amplification of a donor-specific gene. In the present study, we first determined in vitro the sensitivity of standard and nested PCR amplification with sequence-specific primers (PCR-SSP) of a donor-specific allele of the HLA-DRB1 gene and then used this technique to assess prospectively blood chimerism in two single-lung (SLT) and one heart-lung (HLT) transplant recipients. Standard PCR-SSP consisted in a single amplification round with sequence-specific primers for the donor-specific DRB1 allele. Nested PCR-SSP consisted in a first round of generic amplification of exon 2 of the DRB1 gene, followed by a second amplification round with primers specific for the donor allele. In vitro, nested PCR-SSP of the donor-specific allele was 1000-fold more sensitive than standard PCR-SSP and allowed the detection of 1 donor cell in 10(5) recipient cells. In vivo, standard PCR-SSP detected donor cells among the recipients' peripheral blood mononuclear cells (PBMCs) only during the first postoperative days, whereas nested PCR-SSP demonstrated their presence until the end of the first postoperative month in patients 1 and 2 and until 3 months after transplantation in patient 3. We conclude that donor cells can be detected in the peripheral blood of SLT and HLT recipients during the first postoperative months and that nested PCR-SSP amplification of a donor-specific HLA-DRB1 allele is much more sensitive than standard PCR-SSP to demonstrate such chimerism.

Adult↗
PubMed1995

Role of the alpha 1 beta 1 integrin complex in collagen gel contraction in vitro by fibroblasts.

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the beta 1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-beta, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the alpha 2 beta 1 and, to a lesser extent, the alpha 1 beta 1 integrin complexes. The present studies were undertaken to determine the role of the alpha 1 integrin chain, a collagen/laminin receptor, in collagen gel contraction by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the alpha 1 beta 1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the alpha 1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the alpha 1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the alpha 1 beta 1 integrin complex plays a significant role in cellular interactions with interstitial collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing.

Animals↗
PubMed1983

Characterization of a plasma membrane glycoprotein common to myoblasts, skeletal muscle satellite cells, and glia.

A plasma membrane glycoprotein common to embryonic chick myoblasts and adult chicken skeletal muscle satellite cells is the antigen recognized by monoclonal antibody C3/1. Although traces of the same antigen are present on some muscle-derived fibroblasts, the density of antigenic sites on myoblasts and satellite cells is so high that these cell types can be identified in tissues by immunocytochemical techniques. The antigen is exposed on the surfaces of myogenic cells growing in tissue culture and can be solubilized with detergent. This and other criteria establish that the antigen is a plasma membrane protein. The antigen, purified by affinity techniques, consists of a single type of polypeptide chain which migrates as a relatively broad band of apparent molecular weight 38,000 Da in SDS-polyacrylamide gel electrophoresis. It has a very small sedimentation constant, suggesting that the solubilized form is either monomeric or dimeric. The concentration of antigenic sites increases during myogenesis in vitro; but during maturation the antigenic sites are lost from muscle fibers. Electron microscopic autoradiographic study of adult muscle labeled with iodinated monoclonal antibody demonstrated unequivocally that the antigenic sites in adult muscle are concentrated in the satellite cells. Although selective for myoblasts, immature myotubes and satellite cells in the myogenic lineage, the monoclonal antibody also binds at rather high levels to peripheral Schwann cells and teloglia, to some nonneuronal cells in cultures derived from embryonic spinal cord, to some glial elements of adult chicken brain, and to several cell types in the early embryo.

Animals↗
PubMed1988

The cortical reaction in the egg of Discoglossus pictus: a study of the changes in the endoplasmic reticulum at activation.

In Discoglossus pictus previous ultrastructural observations have shown that at the animal dimple, where sperm fuse with the egg, cortical granules (CG), vacuoles, and tightly packed clusters of small cisternae are present. At fertilization the clusters open (i.e., become loose) and give rise to longer cisternae arranged in whorls and chains which migrate toward the plasma membrane. The vacuoles fuse to form cisternae and exocytose along with the CG. In the rest of the egg periphery, while exocytosis occurs, the clusters do not open as a result of activation (C. Campanella, R. Talevi, U. Atripaldi, and L. Quaglia (1986). In "Molecular and Cellular Biology of Fertilization" (J.L. Hedrick, Ed.). Plenum, New York). We have recently conducted electrophysiological studies which have detected inward currents at the dimple center, outward current at the rest of the egg surface, and an eightfold increase in [Ca2+]i which propagates from the site of activation throughout the egg (R. Nuccitelli, D. Kline, W. Busa, R. Talevi, and C. Campanella (1988). Dev. Biol. 130, 120-132). In this paper we have asked whether the anionic current and the Ca2+ increase could be causally related to the changes of the smooth endoplasmic reticulum (SER) at activation. The results obtained by activating the eggs in ion-substituted Ringers indicate that (1) the migration of cisternae is not dependent on the polarity of the activation current crossing the dimple, but is strongly impaired, together with CG exocytosis, by 5 x Cl- Ringer; (2) TMB-8, a drug which partially blocks calcium release (C. Y. Choiu and M. J. Malagodi (1975). Brit. J. Pharmacol. 53, 279-288), partially inhibits opening of cisternae clusters and the formation of an SER network in the dimple. This suggests a causal relationship between the Ca2+ rise and the cluster transformation at activation.

Animals↗
PubMed1988

Laminin, fibronectin and type IV collagen in BM-like material from cultured arterial smooth muscle cells.

1. The intra- and extracellular distribution of fibronectin and laminin was studied by immunofluorescence in cultures of rabbit and human arterial smooth muscle cells. 2. Basement membrane (BM)-like material was isolated from the cell layer of arterial smooth muscle cells cultures and analysed by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) and immunoblotting. The major 220-240 kD component of arterial BM-like material was identified as fibronectin. Also a 200 kD fibronectin band was observed. 3. The 200 kD subunit of laminin was contained in isolated BM-like material, but no slower migrating laminin chains were detected. 4. Collagens were prepared from pepsinized BM-like material. The band pattern as resolved by SDS-PAGE and silver staining suggested that type IV collagen is the major collagen of arterial BM-like material.

Animals↗
PubMed1982

Sulfated glycosaminoglycan and collagen patterns in parietal yolk sac carcinoma (PYSC).

Electrophoretic analyses of collagenous material have shown that the parietal yolk sac carcinoma (PYSC) ascitic tumour synthesizes polypeptide chains that migrate as type IV procollagen. Having molecular weights of 185,000 and 160,000, these polypeptides are sensitive to collagenase. When the PYSC cells are injected subcutaneously, they form a solid tumour, and type I collagen predominates. The electrophoretic analyses of sulfated glycosaminoglycans and enzymatic degradation have shown a predominance of heparan sulfate in the ascitic tumour, and of chondroitin sulfate B in the solid tumour. Cells cultured from ascitic tumours have maintained the same collagen and sulfated glycosaminoglycan patterns as the original cells, whereas in the solid tumour culture only chondroitin sulfate AC has been detected.

Animals↗
PubMed2004

Evidence of newly generated neurons in the human olfactory bulb.

The subventricular zone (SVZ) is known to be the major source of neural stem cells in the adult brain. In rodents and nonhuman primates, many neuroblasts generated in the SVZ migrate in chains along the rostral migratory stream (RMS) to populate the olfactory bulb (OB) with new granular and periglomerular interneurons. In order to know if such a phenomenon exists in the adult human brain, we applied single and double immunostaining procedures to olfactory bulbs obtained following brain necropsy in normal adult human subjects. Double immunofluorescence labelling with a confocal microscope served to visualize cells that express markers of proliferation and immature neuronal state as well as markers that are specific to olfactory interneurons. Newborn cells that express cell cycle proteins [Ki-67, proliferating cell nuclear antigen (PCNA)] were detected in the granular and glomerular layers (GLs) of the human olfactory bulb; these cells coexpressed markers of immature neuronal state, such as Doublecortin (DCX), NeuroD and Nestin. Numerous differentiating cells expressed molecular markers of early committed neurons [beta-tubulin class III (TuJ1)] and were also immunoreactive for glutamic acid decarboxylase (GAD), a marker of GABAergic neurons, or tyrosine hydroxylase (TH), a marker of dopaminergic neurons. Other early committed neurons expressed the calcium-binding proteins calretinin (CR) or parvalbumin (PV). These results provide strong evidence for the existence of adult neurogenesis in the human olfactory system. Despite its relatively small size compared to that in rodents and nonhuman primates, the olfactory bulb in humans appears to be populated, throughout life, by new granular and periglomerular neurons that express a wide variety of chemical phenotypes.

Adult↗
PubMed1998

Carnosine-like immunoreactivity in astrocytes of the glial tubes and in newly-generated cells within the tangential part of the rostral migratory stream of rodents.

In the nervous system, the aminoacylhistidine dipeptide carnosine (beta-alanyl-L-histidine) has been shown to be expressed in the olfactory receptor neurons and in brain astrocytes. Using immunocytochemical techniques, we report here a dense carnosine-like immunoreactivity in the subependymal layer of the rodent forebrain. Since the subependymal layer involves two distinct compartments (astrocytic cells forming glial tubes and newly-generated cells of the rostral migratory stream, here organized to form chains contained within the glial tubes [Brannon Thomas L. et al. (1996) Glia 17, 1-14; Jancovski A. and Sotelo C. (1996) J. comp. Neurol. 258, 112-124; Lois C. et al (1996) Science 271, 978-981; Peretto P. et al. (1997) Brain Res. Bull. 42, 9-21]), we investigated in detail the cellular distribution of carnosine-like immunoreactivity in this area. By using double labelling techniques with antisera raised against carnosine and specific markers of glial tubes or chains of migrating cells, we show that carnosine-like immunoreactivity is associated with both the compartments. On the other hand, unlike markers of the rostral migratory stream, carnosine-like immunoreactivity was not observed in isolated, migrating cells located outside the subependymal layer, which spread through the olfactory bulb in a radially-oriented manner. This suggests that carnosine is transiently expressed by cells of the rostral migratory stream when moving in the tangentially-oriented part of the migration route. Moreover, we investigated the distribution of carnosine-like immunoreactivity in the postnatal rat forebrain and found that it is detectable in the subependymal layer only starting from the third postnatal week, although it is well known that the dipeptide is present in the olfactory receptor neurons since the embryonic day 16 [Biffo S. et al. (1992) J. chem Neuroanat. 5, 5162]. Taken together, these results show that camosine, other than abundantly present in astrocytes of the glial tubes, is associated to the tangential part of the rostral migratory stream.

Animals↗
PubMed1998

Rat mesangial cells express two unique isoforms of laminin which modulate mesangial cell phenotype.

Rat mesangial cells express two unique isoforms of laminin which can be modulated by culture medium composition. To define further the nature of laminin expressed by cultured rat mesangial cells, synthesis of individual laminin chains, as well as their trimeric association, was examined. Based on data from Northern analysis of mRNA expression, immunoblots, immunofluorescence staining and radioimmunoprecipitation of biosynthetically labeled proteins, mesangial cells express laminin beta1, beta2, and gamma1 chains. Mesangial cells do not express laminin alpha1 or alpha2. MC produce a unique alpha chain, designated alpha'm. These laminin chains assemble into two major isoforms. One contains alpha'mbeta1gamma1, co-precipitates with entactin and is assembled into the fibrillar extracellular matrix. The second isoform contains alpha'mbeta2 and a presumed gamma chain that migrates in gel slightly ahead of gamma1. The beta2-containing isoform is concentrated in punctate sites on the cell surface. In addition, mesangial cells display different phenotypes when plated on laminin-1 (alpha1beta1gamma1), as compared to purified beta2. An LRE-containing peptide of laminin beta2 serves as an attachment site for mesangial cells and is sufficient to induce the phenotype observed with intact beta2. These data suggest that laminin isoform expression plays an important role in mesangial cell phenotype and function.

Animals↗
PubMed1975

Electronic structures of cephalosporins and penicillins. 4. Modeling acylation by the beta-lactam ring.

Molecular orbital calculations by the CNDO/2 method are used to study the molecular and electronic details involved in the initial phases of the opening of the beta-lactam ring of a model cephalosporin structure, 7-amino-3-acetoxymethyl-3-cephem. The effect of a simple nucleophile, OH-, approaching the carbonyl carbon center of the beta-lactam ring is monitored by following the charge redistributions that occur in the bicyclic system and in the 3 side chain. A migration of electron density to the ester oxygen of the CH2OAc group is observed with concomitant weakening of the CH2-OAc bond. The results are discussed in relation to the mechanism of acylation of bacterial cell wall enzymes by beta-lactam antibiotics and in relation to the hydrolysis of these molecules. The results indicate that the ability of the 3' substituent of cephalosporins to stabilize electron density transferred to it, i.e., the leavability of the 3' moiety, can be an important factor in activating the beta-lactam toward nucleophilic attack.

Acylation↗
PubMed1997

Biochemical characterization of the arginine-specific proteases of Porphyromonas gingivalis W50 suggests a common precursor.

Extracellular proteases of Porphyromonas gingivalis specific for arginyl peptide bonds are considered to be important virulence factors in periodontal disease. In order to determine the number, inter-relationship and kinetic properties of these proteases, extracellular enzymes with this peptide-bond specificity were purified and characterized from P. gingivalis W50. Three forms, which we denote RI, RI-A and RI-B, accounted for all of the activity in the supernatant. All three enzymes contain an alpha chain of approximately 54 kDa with the same N-terminal amino acid sequence. RI is a heterodimer of non-covalently linked alpha and beta chains which migrate to the same position on SDS/PAGE but which can be resolved by 8 M urea/PAGE. RI-A and RI-B are both monomeric, but the molecular mass of RI-B (70-80 kDa) is significantly increased due to post-translational modification with lipopolysaccharide. All forms show absolute specificity for peptide bonds with Arg in the P1 position and are also capable of hydrolysing N-terminal Arg and C-terminal Arg-Arg peptide bonds. Thus they show limited amino- and carboxy-peptidase activity. For the hydrolysis of Nalpha-benzoyl-L-Arg-p-nitroanilide, the pH optimum is 8.0 at 30 degrees C. The Vmax for all three enzymes is controlled by ionization of two residues with apparent pKas at 30 degrees C of 6. 5+/-0.05 and 9.7+/-0.05, and DeltaH values of approximately 29 kJ/mol and approximately 24 kJ/mol in the enzyme-substrate complex. By analogy with papain, the pKa of 6.5 could be ascribed to a Cys and the pKa of 9.7 to a His residue. E-64 [L-trans-epoxysuccinyl-leucylamide-4-(4-guanidino)butane] is a competitive inhibitor of RI, RI-A and RI-B. Based on physical properties and kinetic behaviour, RI-A appears to be analogous to gingipain from P. gingivalis HG66. However the alpha/beta structure of RI differs significantly from that of the high-molecular-mass multimeric complex of gingipain containing four haemagglutinins described by others. Since the genes for RI and high-molecular-mass gingipain are identical, the data indicate that an alternative processing pathway is involved in the formation of RI from the initial precursor. Furthermore, the identical N-termini and enzymic properties of the catalytic component of RI, RI-A and RI-B suggest that the maturation pathway of the RI precursor may also give rise to RI-A and RI-B. The physiological functions of these isoforms and their role in the disease process may become more apparent through examination of their interactions with host proteins.

Adhesins, Bacterial↗
PubMed1999

Interaction between astrocytes and adult subventricular zone precursors stimulates neurogenesis.

Neurogenesis continues in the mammalian subventricular zone (SVZ) throughout life. However, the signaling and cell-cell interactions required for adult SVZ neurogenesis are not known. In vivo, migratory neuroblasts (type A cells) and putative precursors (type C cells) are in intimate contact with astrocytes (type B cells). Type B cells also contact each other. We reconstituted SVZ cell-cell interactions in a culture system free of serum or exogenous growth factors. Culturing dissociated postnatal or adult SVZ cells on astrocyte monolayers-but not other substrates-supported extensive neurogenesis similar to that observed in vivo. SVZ precursors proliferated rapidly on astrocytes to form colonies containing up to 100 type A neuroblasts. By fractionating the SVZ cell dissociates with differential adhesion to immobilized polylysine, we show that neuronal colony-forming precursors were concentrated in a fraction enriched for type B and C cells. Pure type A cells could migrate in chains but did not give rise to neuronal colonies. Because astrocyte-conditioned medium alone was not sufficient to support SVZ neurogenesis, direct cell-cell contact between astrocytes and SVZ neuronal precursors may be necessary for the production of type A cells.

Adult↗
PubMed2000

Novel negative regulatory element in the platelet-derived growth factor B chain promoter that mediates ERK-dependent transcriptional repression.

Platelet-derived growth factor (PDGF), which consists of an A and/or B chain, stimulates migration and proliferation in vascular smooth muscle cells as well as a large number of other cell types. Investigations over recent years have defined roles for several positive regulatory transcription factors in the PDGF-B promoter. However, little is known about the transcriptional mechanisms that negatively regulate this gene. Here, we used transient transfection and 5' deletion analysis to define a specific region in the PDGF-B promoter-mediating repression in vascular smooth muscle cells. Gel retardation assays revealed this region is bound by nuclear protein(s) in a specific manner. Supershift assays excluded the direct association of Sp1, Sp3, and Egr-1. Mutation of the negative regulatory element no longer supported nucleoprotein complex formation and, when introduced into the PDGF-B promoter, rescued the promoter from repression. Promoter activity was also restored by transfection of oligonucleotide decoys bearing the repressor binding site. The MEK1/2 inhibitor, PD98059, and a dominant negative construct generating inactive ERK1 increased reporter expression driven by the PDGF-B promoter. In contrast, the MEK inhibitor had no effect on the activity of the mutant PDGF-B promoter. These effects were cell type-specific, since neither suppression of the PDGF-B promoter nor nucleoprotein complex formation was observed in vascular endothelial cells. These findings define a distinct negative regulatory element in the PDGF-B promoter that interacts with nuclear protein(s) and inhibits PDGF-B promoter-dependent gene expression in an ERK-dependent manner.

Base Sequence↗
PubMed1991

Regioselective photoreduction of zinc(II) porphyrins to give chlorins.

The ascorbic acid/organic base photoreduction of zinc(II) porphyrins was investigated. It was established that certain substituents can direct the photoreduction to the site of the macrocycle to which they are attached. For example, zinc(II) vinylporphyrins (8, 12, 16, 20) are photoreduced with cis stereochemistry on the ring bearing the vinyl group to give the corresponding chlorins. Zinc(II) acetylporphyrins (22, 24) were likewise reduced to chlorins such that cis-hydrogenation took place on the ring bearing the acetyl group. Zinc(II) formylporphyrins 33 also appear to reduce at the ring bearing the formyl group. When the zinc(II) acrylic porphyrin 28 was photoreduced, reduction did take place at the ring bearing the acrylic side chain, but migration of the acrylate double bond was very rapid, and the product isolated was the corresponding porphyrin propionate 30. Reduction of a zinc(II) porphyrin 35 bearing both a vinyl group and a nuclear carboxylic ester took place at the ring bearing the carboxylic ester. The reaction provides a general method for regioselective synthesis of chlorins from zinc(II) porphyrins without any evidence of formation of over-reduction products characteristic of many other procedures for formation of chlorins from porphyrin precursors.

Metalloporphyrins↗
PubMed1984

Population and social security projections for Bangladesh.

The author presents population projections for Bangladesh using data from the 1981 census, U.N. sources, and other official and nonofficial sources. "Confidence intervals for year 1986 and year 2001 populations for Bangladesh are developed. Rural-urban shifts in population are analyzed through Markov chains, and reveal a significant projected redistribution to urban areas. A study is presented of the actuarial cost of a potential social security program designed to meet the financial needs of an increasing set of urban dwellers.... Aggregate projected contributions and benefits for the years 1975, 2005, 2015, and 2025 illustrate the possible program."

Asia↗