Letter from Poland. Greetings from Warsaw!
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Libraries↗
Biomedical subjects
J Miller
Publications and source records attributed to J Miller.
Alarms are sounded, are we listening?
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Dental Implantation, Endosseous↗
Allograft pancreatic duct dilatation following bladder drained simultaneous pancreas-kidney transplantation: clinical significance.
OBJECTIVE: Radiologic imaging of the allograft pancreatic duct dilatation is an uncommon multifactorial finding that is not well described. The purpose of this investigation is to determine the clinical correlation of this finding. DESIGN: Retrospective study. SETTING: University Hospital, USA. SUBJECTS: One hundred forty five simultaneous pancreas-kidney transplants have been performed for treatment of type I diabetes mellitus and end-stage renal disease between February 1993 and December 1999 at the University of Miami. MAIN OUTCOME MEASURES: In 5 recipients, the pancreatic duct was noted to be dilated by ultrasound 1-18 months post-transplant. RESULTS: In all 5 recipients a Foley catheter was placed as the first line of treatment. This reduced the size of the pancreatic duct in one patient, who presented with normal serum amylase and lipase and hyperglycemia. A pancreas biopsy done with Foley catheter in place showed fibrosis with no evidence of rejection. Four patients with dilated pancreatic duct presented with increased serum amylase and lipase; however improvement of the pancreatic duct dilatation was seen only after anti-rejection therapy. One of the patients developed recurrence of pancreatic duct dilatation and a video-urodynamic study subsequently demonstrated voiding with abdominal straining and no detrusor activity. He ultimately underwent enteric conversion with resolution of pancreatic duct dilatation. CONCLUSIONS: This infrequent complication of dilated transplant pancreatic duct could be multifactorial. It could suggest the diagnosis of reflux pancreatitis, which should resolve with bladder decompression. In fact, 1 patient presented with reflux pancreatitis caused by external sphincter detrusor pseudodyssynergia. However, this ultrasound finding may also be associated with pancreas rejection (4/5 patients).
Adult↗
Using CQI to improve vascular access management in the dialysis unit.
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Catheters, Indwelling↗
Characterisation of the calcium-binding C-terminal domain of Clostridium perfringens alpha-toxin.
Alpha-toxin is the key determinant in gas-gangrene. The toxin, a phospholipase C, cleaves phosphatidylcholine in eukaryotic cell membranes. Calcium ions have been shown to be required for the specific binding of toxin to membranes prior to phospholipid cleavage. Reported X-ray crystallographic structures of the toxin show that the C-terminal domain has a fold that is analogous to the eukaryotic calcium and membrane-binding C2 domains. We report the binding sites for three calcium ions that have been identified, by crystallographic methods, in the C-terminal domain of the protein close to the postulated membrane-binding surface. The position of these ions at the tip of the domain, and their function (to facilitate membrane binding) is similar to that of calcium ions observed bound to C2 domains. Using the optical spectroscopic techniques of circular dichroism (CD) and fluorescence spectroscopy, pronounced changes to both near and far-UV CD and tryptophan emission fluorescence upon addition of calcium to the C-terminal domain of alpha-toxin have been observed. The changes in near-UV CD, fluorescence enhancement and a 2 nm blue-shift in the fluorescence emission spectrum are consistent with tryptophan residue(s) becoming more immobilised in a hydrophobic environment. Calcium binding appears to be low-affinity: Kd approximately 175-250 microM at pH 8 assuming a 1:1 stoichiometry. as measured by spectroscopic methods.
Bacterial Toxins↗
Microangiopathy in kidney and simultaneous pancreas/kidney recipients treated with tacrolimus: evidence of endothelin and cytokine involvement.
BACKGROUND: In the past 3 years, three transplant recipients [one kidney, two simultaneous pancreas/kidney (SPK)] developed a thrombotic thrombocytopenic purpura-like clinical syndrome. This was characterized by an abrupt fall in the hematocrit and platelet count with evidence of hemolysis (fragmented red blood cells and schistocytes) and transplant kidney dysfunction during the first 2 weeks after transplantation. This was also associated with pancreatic dysfunction in the two SPK recipients. In all three patients, elevated tacrolimus levels (>24 ng/ml) occurred. METHODS: Serum cytokine and endothelin levels were determined retrospectively from stored (-70 degrees C) sera. RESULTS: In each case tacrolimus was discontinued, and treatment with plasmapheresis, fresh frozen plasma, steroids, and OKT3 was begun. The clinical courses varied from mild (one patient), to moderate (one patient), to severe (one patient), complicated with seizures and coma. Each patient responded clinically and ultimately was converted to cyclosporine A, and/or mycophenolate mofetil. These clinical events were associated with a rise in serum levels of endothelin and several cytokines. Levels of endothelin were elevated at 209+/-137 pg/ml, particularly in the severe episode where peak levels reached 480 pg/ml (normal 0-20 pg/ml). Peak levels of IL-8 (104+/-36 pg/ml), interleukin- (IL) 10 (238+/-105 pg/ml), and/or IL-12 (306+/-119 pg(ml) mean+/-SE, occurred during or before the clinical event. Serum levels of tumor necrosis factor-a and interferon-gamma were elevated in 2 patients while levels of IL-2, IL-4, and IL-6 were not elevated during the acute phase. CONCLUSIONS: These data are consistent with a mechanism of microangiopathy involving endothelial cell injury (associated with tacrolimus treatment), and accompanied by cytokines (IL-10, IL-12, tumor necrosis factor-a, interferon-gamma) that affect expression of adhesion molecules, chemokines (IL-8) that direct the influx of white blood cells and endothelins that may exacerbate underlying hypertension and increase shear force-related red blood cell destruction.
Adult↗
The human bone marrow as an immunoregulatory organ.
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Animals↗
In vitro immunogenicity of cadaver donor bone marrow cells used for the induction of allograft acceptance in clinical transplantation.
BACKGROUND: The cascade of immunological effects brought about by donor bone marrow cell (DBMC) infusions to induce allograft acceptance in clinical transplantation is not fully understood. Aside from acting as immune responding and regulatory cells, the infused DBMC also may sensitize the recipient to the donor antigens. METHODS: To analyze this stimulatory activity of DBMC, in vitro mixed lymphocyte cultures (MLC) and cell-mediated lymphocytotoxicity (CML) culture systems analogous to the transplant model with DBMC infusion were used. RESULTS: When responding peripheral blood lymphocytes (PBL) from normal volunteers were placed in culture with suspensions of Ficoll-purified, T cell-depleted, un-irradiated allogeneic DBMC (NT-DBMC), a reaction was seen in both MLC and CML. However, when compared to allogeneic spleen cells as stimulating cells, the responses to NT-DBMC were of markedly lower magnitude and were not seen when the NT-DBMC was irradiated (3000 R). When responding PBL were stimulated with either NT-DBMC that had been previously cultured with irradiated cells from the responders for 1 week (activated NT-DBMC), NT-DBMC further depleted of CD15+ and glycophorin A-positive cells (NT-LP/DBMC), or purified CD34+ and CD2+ DBMC subsets, stronger lymphoproliferative and cytotoxic responses were observed. Moreover, these responses were not abrogated by irradiation of the stimulating DBMC subpopulations. Depletion of antigen-presenting cells by positive selection of CD3+ cells from the responding PBL abrogated MLC and CML reactivity, even when purified NT-LP/DBMC, the most stimulatory cells, were used. This latter observation was in contrast to the responses seen with cultures containing allogeneic stimulating spleen cell populations. This indicated the requirement for indirect alloantigen presentation, i.e., the failure of these DBMC to stimulate by direct alloantigen presentation. NT-DBMC was able to stimulate responding PBL in secondary MLC and CML responses with an equivalent magnitude, irrespective of whether the stimulators were spleen cells or NT-DBMC. Finally, the MLC and CML responses were inhibited by tacrolimus (FK506), mycophenolic acid (MPA), and cyclosporine (CsA) in a dose dependent manner, in contrast to previously observed refractoriness of DBMC preparations to these agents if DBMC was tested as responder cells or in modulatory assays. CONCLUSIONS: These results indicate that DBMC are able to function as effective in vitro stimulators, but only by indirect antigen presentation, and that the immune responses mediated by them can be down-regulated by their own inherent suppressive nature, an effect that can be enhanced by the presence of immunosuppressive drugs.
Antibody Formation↗
Use of an antisense strategy to dissect the signaling role of protein-tyrosine phosphatase alpha.
The protein-tyrosine phosphatase PTPalpha has been proposed to play an important role in controlling the dephosphorylation of a number of key signaling proteins and in regulating insulin signaling. To examine the potential cellular functions and physiological substrates of PTPalpha, a potent phosphorothioate oligonucleotide-based antisense strategy was developed that specifically depleted endogenous PTPalpha from 3T3-L1 adipocytes. The antisense probe, alphaAS1, achieved PTPalpha depletion levels normally of >/=85% and which varied up to levels where PTPalpha was not detected at all. Elimination of PTPalpha by 85% inhibited c-Src activity by 80%. Abolishing PTPalpha to levels undetected did not alter the tyrosine dephosphorylation of the insulin receptor or insulin receptor substrate proteins. Moreover, the ability of insulin to activate ERK2 or to stimulate DNA synthesis was not altered by alphaAS1. It is concluded that endogenous PTPalpha is a key regulator of c-Src activity in 3T3-L1 adipocytes and that PTPalpha is not required for the dephosphorylation of the insulin receptor or the insulin receptor substrate proteins or for the regulation of several downstream insulin signaling events in 3T3-L1 adipocytes. Finally, the development of the antisense probe, alphaAS1, provides an important molecular tool of general applicability for further dissecting the roles and precise targets of endogenous PTPalpha.
3T3 Cells↗
A role for the transmembrane domain in the trimerization of the MHC class II-associated invariant chain.
MHC class II and invariant chain (Ii) associate early in biosynthesis to form a nonameric complex. Ii first assembles into a trimer and then associates with three class II alphabeta heterodimers. Although the membrane-proximal region of the Ii luminal domain is structurally disordered, the C-terminal segment of the luminal domain is largely alpha-helical and contains a major interaction site for the Ii trimer. In this study, we show that the Ii transmembrane domain plays an important role in the formation of Ii trimers. The Ii transmembrane domain contains an unusual patch of hydrophilic residues near the luminal interface. Substitution of these polar residues with nonpolar amino acids resulted in a decrease in the efficiency of Ii trimerization and subsequent class II association. Moreover, N-terminal fragments of Ii were found to trimerize independently of the luminal alpha-helical domain. Progressive C-terminal truncations mapped a homotypic association site to the first 80 aa of Ii. Together, these results implicate the Ii transmembrane domain as a site of trimer interaction that can play an important role in the initiation of trimer formation.
Amino Acid Sequence↗
Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems.
Yersinia pestis, the causative organism of plague, produces V antigen (LcrV), a bifunctional protein with regulatory and virulence roles that has been shown to be highly protective against a plague challenge. A combined sub-unit vaccine, comprising recombinant V and Fraction 1 antigens is currently being developed. We report here the expression and purification of recombinant V antigen (rV) using three different expression systems: the N-terminal GST fusion pGEX-5X-2 and pGEX-6P-2 systems from Pharmacia Biotech, and the C-terminal CBD fusion (IMPACT I) system from New England Biolabs. After cleavage from the carrier protein, the yields of rV were 25 mg l(-1) (pGEX-5X-2), 31 mg l(-1) (pGEX-6P-2) and 0.75 mg l(-1) (IMPACT I). All of the recombinant proteins were immunogenic in mice, although there were some differences in their protective efficacy against subcutaneous challenge with Y. pestis. Whilst rV antigen derived from the IMPACT I and pGEX-6P-2 systems and given in two immunising doses protected fully against challenge with 1 x 10(7) colony forming units (cfu) of Y. pestis, there was breakthrough in protection against 1 x 10(5) cfu of Y. pestis in animals immunised twice with rV from the pGEX-5X-2 system. From this study, the pGEX-6P-2 has been selected for the production of rV as a vaccine component. The pGEX-6P-2 system utilises a GST tagged PreScission Protease (a recombinant human rhinovirus 3C protease) to cleave the fusion protein, thereby allowing efficient removal of the enzyme from the final product. In addition, the enzyme is not of animal origin, therefore making it suitable for vaccine production.
Amino Acid Sequence↗
The effect of 17beta-estradiol-DNA adducts on the replication of exon # 5 of the human suppressor gene p53.
Using a PCR technique, exon # 5 of the human tumor suppressor gene p53 was amplified and ligated into the pCRII vector and transformed into Escherichia coli INV alphaF' competent cells. The cloned exon # 5 was 184 bp long. Evidence is presented to show that after dimethyldioxirane epoxidation, 17beta-estradiol was able to form 17beta-estradiol-DNA adducts and to strongly inhibit the replication of the cloned exon # 5 producing smaller sizes of DNA fragments and introducing errors of incorporation at the 3'-end of the terminating DNAs. The errors occurred mainly at the clusters of the complementary 'G' and 'A' bases on the template strand DNA, presumably, the major sites where the 17beta-estradiol-DNA adducts were formed.
Base Sequence↗
XAFS spectroscopy in catalysis research: AXAFS and shape resonances.
Two new techniques in X-ray absorption spectroscopy (XAS) have recently been developed which provide previously unobtainable information on supported noble metal catalysts. Atomic X-ray absorption fine structure (AXAFS) provides direct information on the average changes in interatomic potential of the metal particles induced by metal-support effects. Changes in the interatomic potential can now be directly related to changes in catalytic reactivity. Analysis of shape resonances near the X-ray absorption edge provides information on the changes in the nature of the bonding of adsorbates (like hydrogen) to the metal cluster as induced by the properties of the support. The strong decrease in activity of supported platinum clusters for neo-pentane hydrogenolysis with increasing alkalinity of the support can be ascribed to a decrease in ionization potential of the metal particles directly influenced by the alkalinity of the support.
Journal Article↗
From embryo to adult: persistent neurogenesis and apoptotic cell death shape the lobster deutocerebrum.
Neuronal plasticity and synaptic remodeling play important roles during the development of the invertebrate nervous system. In addition, structural neuroplasticity as a result of long-term environmental changes, behavioral modifications, age, and experience have been demonstrated in the brains of sexually mature insects. In adult vertebrates, persistent neurogenesis is found in the granule cell layer of the mammalian hippocampus and the subventricular zone, as well as in the telencephalon of songbirds, indicating that persistent neurogenesis, which is presumably related to plasticity and learning, may be an integral part of the normal biology of the mature brain. In decapod crustaceans, persistent neurogenesis among olfactory projection neurons is a common principle that shapes the adult brain, indicating a remarkable degree of life-long structural plasticity. The present study closes a gap in our knowledge of this phenomenon by describing the continuous cell proliferation and gradual displacement of proliferation domains in the central olfactory pathway of the American lobster Homarus americanus from early embryonic through larval and juvenile stages into adult life. Neurogenesis in the deutocerebrum was examined by the in vivo incorporation of bromodeoxyuridine, and development and structural maturation of the deutocerebral neuropils were studied using immunohistochemistry against Drosophila synapsin. The role of apoptotic cell death in shaping the developing deutocerebrum was studied using the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling method, combined with immunolabeling using an antiphospho histone H3 mitosis marker. Our results indicate that, in juvenile and adult lobsters, birth and death of olfactory interneurons occur in parallel, suggesting a turnover of these cells. When the persistent neurogenesis and concurrent death of interneurons in the central olfactory pathway of the crustacean brain are taken into account with the life-long turnover of olfactory receptor cells in crustacean antennules, a new, highly dynamic picture of olfaction in crustaceans emerges.
Animals↗
The dependence for leukocyte function-associated antigen-1/ICAM-1 interactions in T cell activation cannot be overcome by expression of high density TCR ligand.
The leukocyte-specific integrin, LFA-1, can enhance T cell activation. However, it is unclear whether the binding of LFA-1 to its ligand, ICAM-1, functions through intercellular adhesion alone, resulting in an augmentation of the TCR signal, or involves an additional LFA-1-mediated cellular signal transduction pathway. We have previously shown that naive CD4+ lymph node T cells, isolated from DO11.10 TCR transgenic mice, are activated by increasing doses of exogenous OVA peptide presented by transfectants expressing both class II and ICAM-1, but not by cells expressing class II alone. To determine whether LFA-1/ICAM-1 interactions were simply enhancing the presentation of low concentrations of specific MHC/peptide complexes generated from exogenously added peptide, we transfected cells with class II that is covalently coupled to peptide, alone or in combination with ICAM-1. These cells express 100-fold more specific class II/peptide complexes than can be loaded onto class II-positive cells at maximum concentrations of exogenous peptide. Despite this high density of TCR ligand, activation of naive CD4+ T cells still requires the coexpression of ICAM-1. LFA-1/ICAM-1 interactions are not required for effective conjugate formation and TCR engagement because presentation of class II/peptide complexes in the absence of ICAM-1 does induce up-regulation of CD25 and CD69. Thus, high numbers of engaged TCR cannot compensate for the lack of LFA-1/ICAM-1 interactions in the activation of naive CD4+ T cells.
Animals↗
Core-binding factor: a central player in hematopoiesis and leukemia.
Consistent chromosomal rearrangements are found in a large number of hematopoietic tumors. In many cases, these rearrangements disrupt genes whose normal function is required for the proper development of blood cells. Excellent examples are the chromosomal rearrangements t(8;21)(q22;q22), t(12;21)(p13;q22), and inv(16)(p13q22) that disrupt two of the genes encoding a small family of heterodimeric transcription factors, core-binding factors (CBFs). CBFs consist of a DNA-binding CBFalpha subunit and a non-DNA-binding CBFbeta subunit. The t(8;21), associated with de novo acute myeloid leukemias, disrupts the CBFA2 (AML1) gene, which encodes a DNA-binding CBFalpha subunit. The t(12;21), the most common translocation in pediatric acute lymphocytic leukemias, also disrupts CBFA2. The CBFB gene, which encodes the non-DNA-binding subunit of the CBFs, is disrupted by the inv(16) in de novo acute myeloid leukemias. All chromosomal rearrangements involving the CBFA2 and CBFB genes create chimeric proteins, two of which have been unequivocally demonstrated to function as transdominant negative inhibitors of CBF function. Both the Cbfa2 and Cbfb genes are essential for normal hematopoiesis in mice, because homozygous disruption of either gene blocks definitive hematopoiesis. Recent data suggest that Cbfa2 and Cbfb are required for the emergence of definitive hematopoietic stem cells in the embryo from a putative definitive hemangioblast precursor. The transdominant negative inhibitor of CBF created by the inv(16), when present from the beginning of embryogenesis, also blocks the emergence of definitive hematopoietic cells in the embryo. On the other hand, chromosomal translocations involving the CBFA2 and CBFB genes in leukemias block hematopoiesis at later steps. This may reflect a difference in the timing at which translocations are acquired in the leukemias, which presumably is subsequent to emergence of the definitive hematopoietic stem cell. The cumulative data suggest that although the earliest requirement for Cbfa2 and Cbfb is for emergence of definitive hematopoietic stem cells, both genes are also required at later stages in the differentiation of some hematopoietic lineages.
Animals↗
Cloning of the human uroplakin 1B cDNA and analysis of its expression in urothelial-tumor cell lines and bladder-carcinoma tissue.
The human uroplakin 1B (UPK1B) gene codes for a structural protein which is a terminal differentiation component of the asymmetric unit membrane on the apical surface of the mammalian bladder. UPK1B is a member of the tetraspan family of proteins, many of which have de-regulated patterns of expression in cancer. Using polymerase-chain-reaction techniques, we have cloned a partial human UPK1B cDNA which codes for the putative full open reading frame for the UPK1B protein. The deduced human UPK1B protein sequence has 92% and 93% amino-acid homology with bovine UPK1b and mink TI1 proteins respectively. Using Northern analysis, we show that the human UPK1B gene is highly expressed in normal human urothelium. However, expression of UPK1B mRNA was undetectable or markedly reduced in 11 out of 16 samples of transitional-cell-bladder-carcinoma tissue and in all 5 bladder-carcinoma cell lines when compared with normal urothelial tissue. The molecular mechanism of down-regulation of RNA expression does not appear to involve gross gene rearrangements or allelic loss.
Amino Acid Sequence↗
TCR, LFA-1, and CD28 play unique and complementary roles in signaling T cell cytoskeletal reorganization.
T cells interacting with APCs undergo rearrangement of surface receptors and cytoskeletal elements to face the zone of contact with the APC. This polarization process is thought to affect T cell signaling by organizing a specialized domain on the T cell surface and to direct T cell effector function toward the appropriate APC. We have investigated the contribution of TCR, CD28, and LFA-1 signaling to T cell cytoskeletal polarization by assaying the response of an Ag-specific Th1 clone toward a panel of transfected APCs expressing MHC class II alone or in combination with ICAM-1 or B7-1. We show that polarization of talin, an actin-binding protein, occurs in response to integrin engagement. In contrast, reorientation of the T cell microtubule-organizing center (MTOC) is dependent on and directed toward the site of TCR signaling, regardless of whether integrins or costimulatory molecules are engaged. MTOC reorientation in response to peptide-MHC complexes is sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. CD28 coengagement overcomes this sensitivity, as does activation via Ab cross-linking of the TCR or via covalent peptide-MHC complexes, suggesting that phosphatidylinositol 3-kinase is not required per se but rather plays a role in signal amplification. Engagement of TCR in trans with LFA-1 results in separation of MTOC reorientation and cortical cytoskeletal polarization events, indicating that the two processes are not directly mechanistically linked. These studies show that T cells mobilize individual cytoskeletal components in response to distinct and specific cell surface interactions.
Actins↗