Biomedical subjects
Publications and source records attributed to T Hoffman.
Mycobacterium tuberculosis infection of the genitourinary tract is an uncommon disease in renal transplant recipients and presentation is atypical. Genitourinary tuberculosis is associated with graft rejection, and this diagnosis should be considered for renal transplant recipients with unexplained fever and constitutional symptoms.
An in vitro model was developed to evaluate the in vivo stability of lanthanide polyaminocarboxylate complexes. The ligand-to-metal ratios for the chelates EDTA, CDTA, DTPA, MA-DTPA (monoamide-DTPA) and DOTA with the lanthanides lanthanum, samarium, and lutetium were optimized to achieve > or = 98% complexation yield for the resultant radiolanthanide complexes. The exchange of the radiolanthanides from their EDTA, CDTA, DTPA, MA-DTPA and DOTA complexes with Ca(2+) was determined by in vitro adsorption and in vitro column studies using hydroxyapatite (HA), an in vitro bone model. In vitro serum stability of these radiolanthanide complexes was used as an additional indicator of in vivo stability, although the mechanism of instability in serum will be different than with bone. The in vitro studies were consistent with the expected findings that the smallest lanthanide (Lu) formed the most stable complexes. In vivo studies were done to validate the in vitro model. Biodistribution studies in normal CF-1 mice showed that in vivo stability of the complex (i.e., the more lanthanide remaining in complex form) could be assessed by a combination of the urinary, bone and liver uptake. For example, biodistribution studies demonstrate that high urinary excretion correlated with complex stability, while high liver plus bone uptake correlated with complex instability. The urinary excretion of the EDTA complexes decreased from (177)Lu to (140)La indicating a loss in stability in the direction of (140)La, consistent with the in vitro studies. The more stable a lanthanide complex is, the lower its exchange with HA in vitro will be, and the lower its combined bone plus liver uptake and higher its urinary excretion will be in vivo. This investigation indicates that the in vivo stability can be determined by a screening method that measures the degree of exchange from the lanthanide chelate with hydroxyapatite (HA) and its serum stability.
We characterized expression and activity of the bile salt transporters Na(+)/taurocholate (TC) cotransporting polypeptide (Ntcp), and bile salt export pump (Bsep), and the expression of organic anion transporting polypeptides 1 and 2 (Oatp1 and 2) and multidrug resistance associated protein-2 (Mrp2) in pregnancy and throughout lactation in rats. The V(max) for Na(+)/TC cotransport in basolateral liver plasma membrane was increased 1.7-fold in 2 days postpartum relative to control and pregnant rats. This correlated well with an increase in Ntcp messenger RNA (mRNA) and a 2-fold increase in Ntcp protein. Ntcp mRNA remained significantly elevated until 14 days postpartum but had begun to decline by 21 days postpartum. The maximal secretory rate (nmol/min/g liver) for TC in the single pass isolated perfused liver was also increased by 10%, 31%, and 24% at 2, 14, and 21 days postpartum and correlated with increased expression of Ntcp and Bsep mRNA and protein. Infusion of ovine prolactin (oPRL) to ovariectomized rats increased expression of both Ntcp and Bsep mRNA and protein. These data indicate a coordinate increased expression of bile salt transporters postpartum and by PRL. Mrp2 mRNA was stable in pregnancy and postpartum, whereas Mrp2 protein expression decreased significantly in pregnancy, but returned to control levels postpartum. Organic anion transporting polypeptide 2 (Oatp2) mRNA was decreased in pregnancy and increased postpartum, but changes in Oatp2 protein were not significant. Oatp1 mRNA and protein were unchanged in pregnancy and postpartum.
The expression of multidrug resistance-associated protein isoform 2 (mrp2), the ATP-dependent export pump that mediates the transport of glucuronic acid-, glutathione-, and sulfate-conjugated derivatives, was studied in rat small intestine. The small intestine was divided into nine equal segments, and mrp2 content was analyzed in homogenate and brush border membrane preparations by Western analysis. mrp2 protein was present mainly in brush border membrane of the proximal segments and gradually decreased from jejunum to the distal ileum. We also analyzed the content of mrp2 in three different populations of proximal enterocytes obtained from the upper and lower villus and the crypt regions. The export pump was mainly expressed in the villus cells and to a lesser degree in the crypt cells of the epithelium. Immunohistochemical analysis performed in duodenum, jejunum, and ileum confirmed in situ the Western blot findings. Analysis of mRNA encoding mrp2 in proximal and distal segments revealed a similar content in both regions, whereas distribution along the villus-crypt axis was similar to the protein gradient. Because conjugating enzymes are distributed similarly to mrp2, we conclude that they may act coordinately to contribute to first-pass metabolism of drugs and other xenobiotics in the proximal small intestine.
Plants commonly respond to pathogen infection by increasing ethylene production, but it is not clear if this ethylene does more to promote disease susceptibility or disease resistance. Ethylene production and/or responsiveness can be altered by genetic manipulation. The present study used mutagenesis to identify soybean (Glycine max L. Merr.) lines with reduced sensitivity to ethylene. Two new genetic loci were identified, Etr1 and Etr2. Mutants were compared with isogenic wild-type parents for their response to different soybean pathogens. Plant lines with reduced ethylene sensitivity developed similar or less-severe disease symptoms in response to virulent Pseudomonas syringae pv glycinea and Phytophthora sojae, but some of the mutants developed similar or more-severe symptoms in response to Septoria glycines and Rhizoctonia solani. Gene-for-gene resistance against P. syringae expressing avrRpt2 remained effective, but Rps1-k-mediated resistance against P. sojae races 4 and 7 was disrupted in the strong ethylene-insensitive etr1-1 mutant. Rps1-k-mediated resistance against P. sojae race 1 remained effective, suggesting that the Rps1-k locus may encode more than one gene for disease resistance. Overall, our results suggest that reduced ethylene sensitivity can be beneficial against some pathogens but deleterious to resistance against other pathogens.
MDR1, an ABC transporter that confers multidrug resistance in tumor cells, is constitutively expressed in normal liver canalicular membrane. Human MDR1-expressing multidrug-resistant cells display increased resistance to estradiol-17beta(beta-D-glucuronide) (E217G). MDR1 substrates/modulators inhibit adenosine triphosphate (ATP)-dependent transport of E217G in the rat canalicular membrane and protect against E217G-mediated cholestasis in isolated perfused rat liver. The present studies were designed to determine if E217G is a substrate for MDR1 using a baculovirus expression system and if other estrogen glucuronides interact with MDR1. ATP-dependent transport of E217G (10 micromol/L) was linear for up to 2 minutes and yielded a rate of 45.6 pmol/min/mg protein in membrane vesicles from Sf9 cells infected with MDR1-baculovirus. This transport was saturable (Km = 62 micromol/L) and occurred into an osmotically sensitive space. ATP-dependent transport of E217G (10 micromol/L) was inhibited 63% by 10 micromol/L daunomycin, but not by 100 micromol/L S-(2,4-dinitrophenyl)glutathione (GS-DNP) (a substrate for canalicular multispecific organic anion transporter [cMOAT]). Glucuronide conjugates of the estrogen D-ring (100 micromol/L), estriol-17beta(beta-D-glucuronide) (E317G) and estriol-16(beta-D-glucuronide) (E316G), inhibited MDR1-mediated E217G transport by 58% and 35%, respectively. In contrast, noncholestatic glucuronides, estradiol-3-(beta-D-glucuronide) (E23G) or estradiol-3-sulfate-17beta(beta-D-glucuronide) (E23SO417G), had no effect. E217G neither stimulated MDR1 ATPase activity nor inhibited verapamil-stimulated ATPase activity. Infusion of 1.5 micromol/L doxorubicin or 1 micromol/L taxol protected against cholestasis induced by E316G and E317G in isolated perfused rat liver. These studies identify E217G, and probably E316G and E317G, as endogenous substrates for MDR1.
beta-Estradiol 17beta-D-glucuronide (E(2)17G), an endogenous cholestatic metabolite of estradiol, has been identified as a substrate for both hepatic P-glycoprotein (P-gp) and the multispecific organic anion transporter (MOAT), the liver-specific homologue of the multidrug resistance protein. The aim of the present studies was to determine the role of hepatic P-gp and MOAT in E(2)17G-mediated cholestasis and its biliary excretion using the isolated perfused rat liver. A bolus dose of E(2)17G (2 micromol) alone decreased the bile flow maximally from 1.5 to 0.3 microl/min/g liver. In the presence of an infusion of 1.5 microM daunorubicin or 1.0 microM Taxol, P-gp substrates, E(2)17G cholestasis was blocked such that 2 micromol E(2)17G decreased the bile flow from 1.48 to 1.31 or from 1.70 to 1.31 microl/min/g liver, respectively. In the presence of 1 and 3 microM Taxol, the log dose-response curves for E(2)17G cholestasis were shifted to the right 2-fold and 5-fold, respectively, in a parallel manner. Taxol (10 and 50 microM) inhibited the ATP-dependent transport of 10 microM E(2)17G in canalicular plasma membrane vesicles by 46 and 81%, respectively. Daunorubicin (1.5 microM) also shifted the log dose-response curve for E(2)17G cholestasis to the right about 4-fold. Neither Taxol nor daunorubicin decreased the biliary excretion of E(2)17G. Infusion of cyclosporine (6 microM), an inhibitor of both P-gp and MOAT, significantly blocked both E(2)17G cholestasis and biliary excretion, such that 16 micromol E(2)17G decreased the bile flow only 15-20%. In contrast, bromosulfophthalein, a MOAT substrate, had no effect on either E(2)17G-mediated cholestasis or its biliary excretion. These data indicate that P-gp plays an essential role in E(2)17G-mediated cholestasis and suggest that MOAT functions to deliver high concentrations of E(2)17G to P-gp.
Translocation-associated Notch homologue (TAN-1), a gene originally cloned from the translocation breakpoint of a human T cell leukemia carrying a 9:7(q34.3) translocation, encodes a protein belonging to the Notch/Lin-12/Glp-1 receptor family. These receptors mediate the specification of numerous cell fates during development in invertebrates and vertebrates. The intracellular portion of Notch/TAN-1 contains six ankyrin repeats that are similar to those found in cytoplasmic I kappa B proteins. I kappa B proteins are specific inhibitors of nuclear factor (NF)-kappa B/Rel transcription factors. Here we show that TAN-1 has functional properties of an I kappa B-like regulator with specificity for the NF-kappa B p50 subunit. A recombinant polypeptide corresponding to the cytoplasmic portion of TAN-1 (TAN-1C) specifically inhibited the DNA binding of p50-containing NF-kappa B complexes. When overexpressed in an appropriate cell line, TAN-1C prevented kappa B-dependent transactivation in transient reporter gene assays in a fashion similar to the structurally related protein, Bcl-3. TAN-1C could activate kappa B-dependent gene expression by attenuating the inhibitory effect of an excess of p50 homodimers. Immunoprecipitation experiments showed that the TAN-1 from a T cell line is associated with NF-kappa B containing p50 and p65 subunits. These observations indicate that TAN-1C may directly engage NF-kappa B transcription factors and modulate nuclear gene expression.
Imipramine is an effective antidepressant agent that blocks the reuptake of monoamines. In order to understand some of its basic mechanisms of action, we investigated the effects of chronic imipramine administration (10 mg/kg, i.p.; 21 days) on the alpha-2 receptor population of several brain sites. Alpha-2 receptor density was estimated by in vitro autoradiography using [3H]Idazoxan. The densitometric analysis revealed a decreased receptor density in the A2 region of the tractus solitarius (20%) and locus coeruleus (16%). No changes were observed in the amygdala, pyriform cortex, periacueductal gray and the bed nucleus of the stria terminalis. These results suggest that chronic imipramine treatment selectively modulates the alpha-2 receptor population localized in the brain stem norepinephrine-rich nuclei and not in the population present on limbic structures innervated by noradrenergic terminal projections. The possible physiological consequences of this selective modulation of alpha-2 receptors are discussed.
The ATP-dependent transport of beta-estradiol 17-(beta-D-glucuronide) (E217G), a cholestatic metabolite of estradiol, was investigated in rat liver canalicular membrane vesicles. ATP-dependent transport was dependent on time and temperature and occurred into an osmotically sensitive space; kinetic analysis indicated a saturable transport system (Michaelis-Menten constant value, 75 microM; maximum transport rate, 598 pmol.min-1.mg protein-1). The steroid conjugates estradiol glucuronide, estriol 3-glucuronide, estriol 16 alpha-glucuronide, testosterone glucuronide, and the three-sulfate conjugate of 17G were effective inhibitors of transport. Bromosulfophthalein, S-(2,4-dinitrophenyl)glutathione, and glutathione disulfide, all substrates of the canalicular ATP-dependent non-bile acid organic anion transport system, were also effective inhibitors, whereas taurocholate had no effect on transport. Conversely, E217G inhibited the ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione. Daunorubicin, vinblastine, etoposide, cyclosporin, and PSC-833, substrates/modulators of P-glycoprotein, were also potent inhibitors of E217G transport, and E217G competitively inhibited the ATP-dependent transport of daunorubicin. C219, a monoclonal antibody against P-glycoprotein, inhibited ATP-dependent transport of E217G and daunorubicin but not of taurocholate or S-(2,4-dinitrophenyl)glutathione. These data indicate that E217G is substrate of both the non-bile acid organic anion transport system and P-glycoprotein but not of the ATP-dependent bile acid transport system in canalicular membranes.
Explore the source record for details and available documents.
dlk encodes a transmembrane protein member of the EGF-like family of homeotic proteins. dlk is expressed in the same type of neuroendocrine tissues and tumors as pG2, a gene cloned because of its differential expression in human pheochromocytomas versus neuroblastomas. Human dlk and pG2 cDNAs are around 98% similar in sequence, but the predicted proteins encoded by those genes are apparently unrelated. This fact suggested the existence of polymorphic variants of the same gene. We have sequenced again several pG2 and dlk clones in parallel. We identified a pG2 cDNA species corresponding to an alternatively spliced dlk mRNA, as well as several other variant forms of dlk mRNA. One of the pG2 clones resulted to be identical to human dlk and encode the same EGF-like protein. Pref-1, a cDNA isolated from 3T3-L1 fibroblasts, encodes a putative protein possessing an extracellular EGF-like domain similar to dlk, but a different intracellular region. Analysis of sequence data from different clones obtained in our laboratory confirmed some of the differences between dlk and Pref-1. However, the putative difference in the intracellular regions of dlk and Pref-1 was due to sequence artifacts. These data suggest that dlk, pG2 and Pref-1 are variant products of the same gene.
OBJECTIVES: We report on the implementation of digital processing in a large clinical and research sleep laboratory. The system includes the digital collection, display, analysis, and repository of physiological signals collected during sleep. METHODS: After describing the original analog system, the computer equipment and software necessary for the digital implementation are presented and we explain our algorithms for rapid eye movement (REM) and delta-wave detection. Finally, we describe an experiment validating the digital system of display and analyses. CONCLUSIONS: The digital processing of sleep signals saves computer operator, polysomnographic technologist, and computer time. It also saves resources such as polysomnographic paper and FM tape. The digital signals lend themselves to a large array of analysis techniques and result in improved signal quality. Automated REM and delta-wave detection via digital processing correlate highly with visual counts of rapid eye movements and delta waves.
Improvement of antibody responses to polysaccharides through their linkage to proteins is thought to be mediated by protein-specific T helper (Th) cells. To investigate whether the carrier protein of a conjugate could be substituted by a Th epitope, Streptococcus pneumoniae type 17F polysaccharide (PS) was bromoacetylated and coupled to different peptides via their carboxy-terminal cysteines. Two peptides, one from the mycobacterial 65-kDa heat shock protein (hsp65) and the other from influenza virus hemagglutinin, are well-known Th epitopes. Two other peptides were selected from the pneumolysin sequence by Th epitope prediction methods; one of them was synthesized with cysteine either at the carboxy or the amino terminus. Three conjugates consistently elicited in mice anti-PS immunoglobulin M (IgM) and IgG responses that were not observed upon immunization with derivatized PS without peptide. The same conjugates induced no anti-PS antibody responses in athymic (nu/nu) mice, whereas clear responses were elicited in euthymic (nu/+) controls, demonstrating the thymus-dependent character of these conjugates. Only the three conjugates inducing anti-PS responses were capable of eliciting antipeptide antibodies. One of the immunogenic conjugates was studied in more detail. It induced significant protection and an anti-PS IgG response comprising all subclasses. On the basis of these results and proliferation studies with peptide and conjugate-primed cells, it is concluded that linkage of Th epitopes to PS in the right orientation enhances its immunogenicity in a thymus-dependent manner. Future possibilities for using peptides as carriers for inducing antibody responses to poorly immunogenic saccharide antigens are discussed.
The latent membrane protein 1 (LMP1) oncogene is one of the major proteins synthesized by the Epstein-Barr virus (EBV). It is expressed in Reed-Sternberg cells of Hodgkin's disease (HD), tumor cells of nasopharyngeal carcinoma (NPC), and immunoblasts of angioimmunoblastic lymphadenopathy (AILD). A particular LMP1 deletion mutant was recently identified in NPC and clinically and histologically aggressive HD. We studied two patients with AILD that subsequently progressed into immunoblastic lymphoma (IBL) in order to investigate whether the LMP1 deletion mutant was implicated in progression of AILD into IBL. Immunohistology and in situ hybridization were performed on diagnostic biopsies. DNA extracted from fresh frozen material was used for rearrangement studies and polymerase chain reaction (PCR) based amplification and sequencing of portions of the LMP1 gene. Immunohistochemistry revealed B cell origin of both cases of IBL. In the first patient clonal rearrangement of the immunoglobulin heavy-chain gene was present in IBL but not in AILD. In this patient, scattered immunoblasts of AILD and numerous tumor cells of B-IBL were shown to contain EBV transcripts (EBER1) and to express LMP1. Sequence analysis of the LMP1 gene from AILD and IBL in the first, and from IBL in the second patient, revealed identical deletions and point mutations. This LMP1 deletion mutant is identical to those which have been reported in HD and NPC. Its association with evolution of AILD into B-IBL, aggressive HD and NPC, suggests that this particular mutant is more widespread than originally thought and is clinically relevant.
We recently reported that a significant population of the murine splenic non-T, non-B "null" cell compartment consists of non-lineage-specific, undifferentiated cells which are in the G0 and G1 phases of the cell cycle and that their numbers are particularly high in the spleens of New Zealand Black mice. A highly enriched population of these non-lineage-specific cells obtained by successive elimination of differentiated cells was further purified to homogeneity by fluorescence-activated cell sorting. The morphologic, phenotypic, and histochemical characteristics of this purified population suggest that these cells may be primitive hematopoietic stem cells. The germ line configuration of the genomic DNA establishes that these are uncommitted stem cells. In vivo, these cells form day 12 colonies in the spleen and liver of lethally irradiated recipients and confer radioprotection. These cells also differentiate into T- and B-cell lineages and reconstitute the immunodeficiency in mice with severe combined immunodeficiency. In response to a combination of a very few early-acting lymphokines and/or stromal cell-conditioned medium in vitro, these cells differentiate into both myeloid and lymphoid cell types. More of these cells are obtained from the enlarged spleens of New Zealand Black mice than from those of BALB/c mice. The presence of a comparatively higher number of stem cells in the spleen than in the marrow or fetal liver provides an alternative, and possibly superior, source of uncommitted stem cells for a variety of experimental investigations or therapeutic manipulations.
Estradiol-17 beta-glucuronide (E(2)17G) is one of a series of naturally occurring glucuronide conjugates of the steroid D-ring that induce cholestasis in the rat and nonhuman primate. The present studies characterized the transport of [3H]E(2)17G in basolateral membrane vesicles (BMVs) from male rat liver. The uptake of E(2)17G was temperature dependent, occurred into an osmotically sensitive space, and was saturable, with an apparent Michaelis constant and maximal velocity of 13 microM and 29 pmol.mg protein-1.5 s-1, respectively. Uptake was not coupled to the uptake of Na+ nor to Cl-or OH- exchange but was markedly stimulated by an inside-positive membrane potential. Bromosulfophthalein inhibited the uptake of E217G noncompetitively; estriol-16 alpha-glucuronide, but not estradiol-3-glucuronide or estradiol-3-SO4-17 beta-glucuronide, inhibited the uptake of E(2)17G. Thus E(2)17G is transported as an anion by facilitated diffusion by a system that differs from the multispecific Na(+)-taurocholate cotransport system and the bromosulfophthalein transport system.
The multidrug resistance (MDR) gene family has been shown to be highly expressed in several normal tissues including the canalicular membrane of the hepatocyte. We report that a cholestatic estrogen metabolite, 17 beta-estradiol glucuronide (E217G), is a substrate for the MDR transporter, P-glycoprotein. In cytotoxicity studies, the MDR sarcoma cell line Dx5 was 4.7-fold resistant to E217G, and the K562/R7 leukemia MDR cell line was 5.0-fold resistant to E217G relative to their parental cell lines. There was also a 2- to 3-fold accumulation defect of [3H]E217G in the MDR cells relative to their parental cell lines. E217G (100 microM) modulated resistance ot doxorubicin, taxol, vinblastine, and etoposide in the Dx5 cells, completely reversing the 30- to 60-fold resistance observed with these agents. E217G had no effect on the toxicity of these compounds in the parental cell line (MES-SA). In contrast, MDR cells were not resistant to the noncholestatic estrogen metabolite, estriol 3-glucuronide, and this metabolite did not modulate resistance to MDR substrates. ATP-dependent transport of [3H]E217G in rat canalicular membranes was inhibited by several MDR substrates including vinblastine, etoposide, verapamil, cyclosporine, and PSC-833.