Correlation between changes in cellular immunity and rejection in rat small bowel transplantation.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
X He
Publications and source records attributed to X He.
Rabbit plasma, unlike its human counterpart, contains no complex between protein S and C4b-binding protein.
In human plasma, the anticoagulant vitamin K-dependent protein S exists in two molecular forms, as free protein and complexed to C4b-binding protein (C4BP), a complement regulatory protein. It has been suggested that rabbit plasma also contains two forms of protein S and that the interaction between protein S and C4BP in rabbits can be modulated by synthetic peptides corresponding to a sequence (residues 605-614) in the carboxy-terminal part of protein S. In this report, we provide results which challenge the conclusion that rabbit plasma contains the complexed form of protein S. The two forms of protein S in human plasma were separated by gel filtration chromatography on Sephacryl S-300 and the presence of protein S in the various fractions analyzed by Western blotting using a monoclonal antibody (HPS 21) directed against the gamma-carboxyglutamic acid rich module of human protein S. This antibody, which was found to cross-react with rabbit protein S on Western blotting, was used in affinity purification of protein S from rabbit plasma as well as of recombinant rabbit protein S. HPS 21 specifically recognized protein S in rabbit plasma and did not cross-react with the other vitamin K-dependent plasma proteins. To elucidate whether rabbit plasma contained two forms of protein S, rabbit plasma was subjected to gel-filtration chromatography followed by Western blotting of the fractions with monoclonal antibody HPS 21. Protein S was found only in fractions eluting at a position corresponding to that of free protein S.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Integration and expression of human growth hormone gene in Caladium bicolor.
Human growth hormone (hGH) gene has been inserted into the plasmid pLGV1103 to give the recombinant plasmid pLB-9. It has been introduced into the agrobacterium containing plasmid pGV3850. The recombinant Ti plasmid pGL198(hGH) has been obtained by homologous recombination. The monocotyledon Caladium bicolor has been transferred with pGL198 (hGH) with the leaf-disk co-cultivation method, and transgenic plants have been regenerated. The results of nopaline analysis, NPT II detection Southern blot and Western blot show that the hGH gene was integrated into the genome of Caladium bicolor, and a 22-kD protein was synthesized in the transgenic plants.
Gene Transfer Techniques↗
Further studies of the structure-activity relationships of 1-[1-(2-benzo[b]thienyl)cyclohexyl]piperidine. Synthesis and evaluation of 1-(2-benzo[b]thienyl)-N,N-dialkylcyclohexylamines at dopamine uptake and phencyclidine binding sites.
We previously reported (J. Med. Chem. 1993, 36, 1188-1193) that changes to the ring size of the piperidine and cyclohexyl rings of the high-affinity and selective dopamine (DA)-uptake inhibitor 1-[1-(2-benzo[b]thienyl)cyclohexyl]piperidine (BTCP, 2) caused different, and in some cases opposite, changes in affinity for sites on the DA transporter labeled by [3H]BTCP and [3H]-cocaine. These results suggested that the radioligands label different sites on the transporter. In the present study, we extend the structure-activity relationships (SAR) of BTCP by studying the binding characteristics of a series of N,N-disubstituted 1-(2-benzo[b]thienyl)cyclohexylamines 7-32 at the DA transporter. Cyclohexyl was selected as opposed to other ring sizes since it corresponds to BTCP. The binding results indicate that a considerable degree of structural variation is permitted for the N-substituents, while still retaining nanomolar affinity for sites on the transporter (studied in rat forebrain homogenates). As observed in our earlier study, the differential effects of structural change on binding to sites on the DA transporter labeled by these radioligands suggests that they are different and distinct binding sites. In general, and up to a point, increasing the size and lipophilicity of the N substituents resulted in improvements in binding but appeared to have less predictable effects on DA-uptake inhibition (as measured in rat brain synaptosomes). The binding of these compounds to sites labeled by [3H]BTCP appeared to correlate best with IC50 for DA-uptake inhibition. To our surprise, the monoalkyl N-substituted BTCP derivatives displayed the highest affinity for the DA transporter of all the compounds in this series. For example, the N-(cyclopropylmethyl) derivative 14 displayed IC50's = 23 nM ([3H]cocaine) and 1 nM ([3H]-BTCP), and the N-butyl derivative 10 showed IC50's = 60 nM ([3H]cocaine) and 0.3 nM ([3H]-BTCP). BTCP exhibited IC50's of 39 nM ([3H]cocaine) and 5 nM ([3H]BTCP) in this assay. The observation that N,N-dibutyl derivative 31 exhibited low ratios of IC50 [3H]cocaine/IC50 DA reuptake and IC50 [3H]BTCP/IC50 DA reuptake suggests that it may be a potential candidate for cocaine antagonism studies. The effect of additional amino, amide, and aromatic groups on the N-substituents was examined, and the results are discussed. The failure of all of the compounds in this series to bind phenycyclidine receptors coupled with their high affinity and range of selectivities at the DA transporter identifies many of them as useful tools for probing the mode of action of BTCP at this site.
Animals↗
Molecular cloning, expression and functional characterization of rabbit anticoagulant vitamin-K-dependent protein S.
Vitamin-K-dependent protein S is an anticoagulant plasma protein which functions as cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa. In addition, it interacts with C4b-binding protein (C4BP), a regulator of the complement system. Using a human protein S cDNA clone as probe, cDNA clones for rabbit protein S were isolated from a rabbit liver cDNA library. The cDNA sequence encoded the mature protein S and 12 residues of the leader sequence. The amino acid sequence of the single-chain 634-amino-acid-residue-long rabbit protein S molecule was 82% and 81% identical to those of human and bovine protein S, respectively. Northern blotting demonstrated protein S mRNA not only in liver but also in reproductive organs (testis, ovary and uterus), in lung and brain. Recombinant rabbit protein S was expressed in eucaryotic cells and found to be post-translationally modified, i.e. it had the correct amino terminus, contained N-linked carbohydrate side chains, gamma-carboxyglutamic acid residues and beta-hydroxylated aspartic acid and asparagine residues. Recombinant rabbit protein S bound calcium like its human counterpart, as judged by its migration in the presence of calcium on agarose-gel electrophoresis. Rabbit protein S has been reported to be species specific with respect to its interaction with APC and not to function with bovine APC. However, we found it to act as cofactor to both human and bovine APC, albeit it was somewhat more efficient with human than with bovine APC. Rabbit protein S, like its human and bovine counterparts, bound human C4BP in a reaction which was associated with the loss of its APC-cofactor activity. However, unlike human plasma, rabbit plasma appeared to contain only the free form of protein S as a radiolabeled rabbit protein S tracer added to rabbit plasma migrated as free protein S on agarose-gel electrophoresis. Addition of human C4BP to rabbit plasma resulted in the formation of a C4BP-protein-S complex, suggesting an explanation for the absence of complexed protein S in rabbit plasma to be sought for in the structure of rabbit C4BP.
Amino Acid Sequence↗
Aortic smooth muscle contains guanylate-cyclase-coupled 130-kDa atrial natriuretic factor receptor as predominant receptor form. Spontaneous switching to 60-kDa C-receptor upon cell culturing.
Photoaffinity labeling of atrial natriuretic factor (ANF) receptor in the plasma membranes from bovine aortic smooth muscle tissue using N alpha 5-(4-azidobenzoyl)-ANF-(5-28)- peptide labeled with 125I yielded a 130-kDa band. However, when smooth muscle cells from the same bovine aorta were placed in culture, the 130-kDa receptor quickly disappeared and a 60-kDa band began to appear at high density. After three passages, essentially no 130-kDa band was found and only the 60-kDa band was strongly labeled. The primary structures of the two receptor forms were compared by radiochemical peptide mapping after endoproteinase Glu-C digestion of photoaffinity-labeled and detergent-solubilized 130-kDa receptor from the aorta or the 60-kDa receptor from the cultured cells. The peptide mapping showed courses of digestion that were significantly different from each other, suggesting difference in their primary structures. The basal guanylate cyclase activity in the aortic membranes was 1.0 pmol cGMP produced.min-1.mg protein-1 at 37 degrees C using Mn(2+)-GTP as substrate. The corresponding activity in the membranes from the cultured cells was 20 fmol cGMP.min-1.mg protein-1. Binding studies gave a density of binding sites (Bmax) of 82 fmol/mg protein for the aortic membranes and 850 fmol/mg protein for the cultured cell membranes. These data suggest that the major form of ANF receptor in the cultured cells, namely the 60-kDa receptor, lacked guanylate cyclase activity. Northern blot analysis of poly(A)-RNA extracted form bovine thoracic aorta or adrenal cortex gave a single 3.6-kb band when 32P-labeled human A-type ANF receptor cDNA was used as a hybridization probe. However, no band was detected when C-receptor cDNA was used as a probe. In addition to the major 130-kDa band, extended SDS/PAGE revealed two additional faint bands with estimated molecular masses of 126 kDa and 135 kDa. Treatment with endoglycosidase H resulted in disappearance of the 126-kDa band and appearance of a 100-kDa band. The 130-kDa and 135-kDa bands were unchanged. Treatment by endoglycosidase F or glycopeptidase F reduced all three bands to a single 100-kDa band. These results suggest that the slight difference in mobility is due to different states of glycosylation.(ABSTRACT TRUNCATED AT 400 WORDS)
Affinity Labels↗
Magnetism of Al-Mn quasicrystals.
Explore the source record for details and available documents.
Journal Article↗
The repertoire of rheumatoid factor-producing B cells in normal subjects and patients with rheumatoid arthritis.
OBJECTIVE: To compare the B cell repertoire of normal individuals and patients with rheumatoid arthritis (RA) and, specifically, to identify precursor B cells with the potential to secrete rheumatoid factor (RF) and to understand the T helper cell requirements for the production of this autoantibody. METHODS: Frequencies of precursors of IgM-, IgG-, and RF-producing B cells were measured in a limiting-dilution system. Two distinct sources of T cell help were compared. T cell help was provided by anti-CD3-activated CD4+ human T cell clones, or T cell-B cell interaction was facilitated by the bacterial super-antigen staphylococcal enterotoxin D (SED). RESULTS: A subset of 2-14% of peripheral blood B cells secreted IgM and IgG in SED-driven cultures. The SED-responsive B cell subpopulation was present at 10 times higher frequency in normal donors compared with RA patients. However, the repertoires were very similar, particularly for RF+ precursors, which represented approximately one-third of all SED-responsive B cells. In normal individuals, most of these RF+ precursor B cells did not respond to anti-CD3-activated T helper cells, with only a very small fraction of B cells activated by anti-CD3-driven helper cells maturing into RF-secreting B cells (from 1 of 182 to 1 of 889 IgM-producing B cells). This subset was expanded approximately 50-fold in RA patients. CONCLUSION: Normal subjects and RA patients share a pool of B cells which secrete RF when activated in the presence of SED and T helper cells. These B cells are frequent and obviously anergic in normal individuals. The B cell subset with the potential to produce RF when help is provided in noncognate T-B interaction (anti-CD3-driven T cells) is considerably expanded in RA patients, probably reflecting an increased responsiveness of such B cells to helper signals.
Arthritis, Rheumatoid↗
Assessment of the antibody response to the immunosuppressive/immunodominant region of HIV gp41 in a 5-year longitudinal study.
The antibody response of HIV-infected individuals to the 581-609 amino acid (aa) region of HIV-1 gp41 containing the putative immunosuppressive and immunodominant sequences was examined. Sera collected every 5 to 6 months over a period of 5 years from 50 HIV-1-infected homosexual and bisexual men, 25 of whom progressed to AIDS during the collection period, were monitored for changes in ELISA reactivity against synthetic peptides encompassing aa581-609 of gp41. The data obtained in this blinded, historical prospective study were analyzed with respect to changes in mean ELISA absorbance over time and differences in absorbance between patient groups (those who progressed to AIDS and those who did not). No correlation was found between time or disease state and the presence of antibodies to the aa581-597 immunosuppressive sequence. In contrast, ELISA absorbance against the aa598-609 immunodominant sequence continued to increase over time in both the AIDS and non-AIDS groups. The rate of increase in absorbance was similar for both groups; however, the AIDS group had a significantly higher mean absorbance level at the outset of the study and maintained this difference throughout the study. This region of gp41 has been proposed to play a role in complement-mediated antibody-dependent enhancement of infection.
Acquired Immunodeficiency Syndrome↗
Simple calculation of the velocity profiles for pulsatile flow in a blood vessel using Mathematica.
Explore the source record for details and available documents.
Algorithms↗
Attempts to eradicate methicillin-resistant Staphylococcus aureus from a long-term-care facility with the use of mupirocin ointment.
PURPOSE: To assess the impact of the use of mupirocin ointment on colonization, transmission, and infection with methicillin-resistant Staphylococcus aureus (MRSA) in a long-term-care facility. PATIENTS AND METHODS: All 321 residents of a Veterans Affairs long-term-care facility from June 1990 through June 1991 were studied for MRSA colonization and infection. MRSA-colonized patients received mupirocin ointment to nares in the first 7 months and to nares and wounds in the second 5 months. The effect of mupirocin use on MRSA colonization and infection was monitored. All S. aureus strains isolated were tested for the development of resistance to mupirocin. RESULTS: A total of 65 patients colonized with MRSA received mupirocin ointment. Mupirocin rapidly eliminated MRSA at the sites treated in most patients by the end of 1 week. Weekly maintenance mupirocin was not adequate to prevent recurrences--40% of patients had recurrence of MRSA. Overall, MRSA colonization in the facility, which was 22.7% +/- 1% prior to the use of mupirocin, did not change when mupirocin was used in nares only (22.2% +/- 2.1%), but did decrease to 11.5% +/- 1.8% when mupirocin was used in nares and wounds. Although colonization decreased, roommate-to-roommate transmission and MRSA infection rates, low to begin with, did not change when mupirocin was used. Mupirocin-resistant MRSA strains were isolated in 10.8% of patients. CONCLUSIONS: Mupirocin ointment is effective at decreasing colonization with MRSA. However, constant surveillance was required to identify patients colonized at admission or experiencing recurrence of MRSA during maintenance treatment. Long-term use of mupirocin selected for mupirocin-resistant MRSA strains. Mupirocin should be saved for use in outbreak situations, and not used over the long term in facilities with endemic MRSA colonization.
Aged↗
The isolation and structural elucidation of four novel triterpene lactones, pseudolarolides A, B, C, and D, from Pseudolarix kaempferi.
Four novel triterpene lactones, pseudolarolides A [1], B [2], C [3], and D [4], were isolated from the seeds of Pseudolarix kaempferi. Their structures and stereochemistry were elucidated from spectral data. Compound 2 shows potent cytotoxicity against three human cancer cell lines, KB (nasopharyngeal), A-549 (lung), and HCT-8 (colon), and against a murine leukemia cell line (P-388) with ED50 values of 0.49, 0.67, 0.73, and 0.79 micrograms/ml, respectively.
Animals↗
Spacing and orientation of bipartite DNA-binding motifs as potential functional determinants for POU domain factors.
Investigation of the large POU domain family of developmental regulators has revealed a molecular mechanism by which highly related transcription factors sharing common DNA-binding motifs act to functionally discriminate their cognate DNA sequences. Studies of two classes of neuron-specific POU domain factors (III and IV) indicate that functional specificity on their native response elements is achieved by accommodating different nucleotide spacing between variably oriented bipartite core DNA-binding motifs. The preferred orientation of the POU-specific domain of the neuronal factors on their native response elements appears to be opposite that of Pit-1 and Oct-1. Members of POU-III (Brn-2) class exhibit remarkable flexibility in DNA site recognition (tolerating core motifs spaced by 0, 2, or 3 nucleotides), whereas POU-IV (Brn-3) class is highly constrained (tolerating core motifs with a spacing of 3 nucleotides). The molecular determinant of the constraint in DNA site selection appears to be imparted by 3 amino acid residues in the amino-terminal basic region in concert, with helix 2 of the POU homeo domain which together are involved in minor groove and possibly phosphate backbone contacts. Similar mechanisms may underlie differential flexibility in spacing and orientation for diverse families of transcription factors.
Amino Acid Sequence↗
Numerical study of the quasi-Landau resonances of sodium.
Explore the source record for details and available documents.
Journal Article↗
Hydrogenic circular states in a superstrong magnetic field: A B-spline approach.
Explore the source record for details and available documents.
Journal Article↗
Functional expression of the rat anion exchanger AE2 in insect cells by a recombinant baculovirus.
We used baculovirus to transiently express a rat anion exchanger (AE2) in Spodoptera frugiperda (Sf9) insect cells. No detectable Cl(-)-HCO3- exchange activity was observed in wild type or sham-infected Sf9 cells, monitored using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, a pH-sensitive fluorescent dye. Functional expression of anion exchange activity in the AE2 recombinant baculovirus-infected cells was observed within the first day after infection and sustained over the next 3 days. The expressed AE2 anion exchange activity was Na+ independent and could be reversibly and irreversibly inhibited by the specific anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). The reversible inhibition was sensitive to the concentration of DIDS, with a half inhibition of 4 microM. These results indicate that the rat AE2 protein produced in the recombinant baculovirus-infected insect cells is inserted into the plasma membrane in a biologically active form that appears suitable for functional studies of AE2.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Plasminogen activator inhibitor-1 mRNA is expressed in platelets and megakaryocytes and the megakaryoblastic cell line CHRF-288.
Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet alpha-granule and is released on platelet activation. Platelet PAI-1 could either be synthesized by the megakaryocyte or taken up from the plasma. In this report we confirm the presence of PAI-1 protein in human megakaryocytes by Western blot analysis and show its synthesis in guinea pig megakaryocytes by metabolic labeling. We document the presence of PAI-1 mRNA in human platelets and show a 3-kb mRNA species on Northern blot analysis of guinea pig megakaryocytes. Neither untreated CHRF-288 cells, a megakaryoblastic cell line, nor human erythroleukemia (HEL) cells expressed PAI-1 mRNA. Phorbol ester (phorbol 12-myristate 13-acetate, 160 nM) treatment of CHRF-288 and HEL cells for 4 days induced PAI-1 mRNA expression in CHRF-288 cells but not in HEL cells. These studies show that PAI-1 is synthesized by megakaryocytes. Megakaryocytes most likely determine the PAI-1 content of platelets and thereby establish the antifibrinolytic potential of the platelet.
Animals↗
P-selectin mRNA is expressed at a later phase of megakaryocyte maturation than mRNAs for von Willebrand factor and glycoprotein Ib-alpha.
The assembly of alpha-granules occurs exclusively in megakaryocytes because platelets have limited capacity for the synthesis of macromolecules. Thus far, alpha-granule development in megakaryocytes has been primarily evaluated by ultrastructural studies. The aim of the study was to obtain molecular and biochemical evidence for the expression of selected alpha-granule proteins in megakaryocytes. Guinea pig megakaryocytes were purified and separated into subgroups at different phases of maturation by the Celsep procedure (Schick et al. Blood 1989;73:1801-8). Guinea pig-specific probes for P-selectin, von Willebrand factor (vWF), glycoprotein Ib-alpha (GpIb-alpha), and phosphoglycerate kinase were prepared by using the polymerase chain reaction. By Northern blot analysis, P-selectin messenger ribonucleic acid (mRNA) was primarily expressed in the mature megakaryocyte Celsep subgroup, whereas vWF and GpIb-alpha mRNA were expressed at all phases of megakaryocyte maturation. In situ hybridization confirmed that P-selectin mRNA was primarily expressed at later stages of cytoplasmic maturation: 14% +/- 6.2% of stage I, 35.5% +/- 6.1% of stage II, 72% +/- 5.2% of stage III, and 47.0% +/- 3.3% of stage IV megakaryocytes expressed P-selectin mRNA. Thus, the expression of mRNA for P-selectin appeared to peak in stage III cells. In contrast vWF mRNA was expressed in immature megakaryocytes and persisted throughout megakaryocyte maturation. In situ hybridization did not demonstrate a relationship between the expression of mRNA for P-selectin or vWF with megakaryocyte ploidy.(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of Variance↗