Salmonella-induced apoptosis of infected macrophages results in presentation of a bacteria-encoded antigen after uptake by bystander dendritic cells.

Salmonella typhimurium is a gram-negative bacterium that survives and replicates inside vacuolar compartments of macrophages. Infection of macrophages with S. typhimurium grown under conditions allowing expression of the type III secretion system results in apoptotic death of the infected cells. Here, we show that infection of bone marrow-derived macrophages (MPhi) with wild-type S. typhimurium 14028 results in presentation of epitopes derived from a bacteria-encoded antigen on major histocompatibility complex (MHC) class I and MHC class II molecules after internalization of apoptotic MPhi by bystander dendritic cells (DCs). In contrast, infection of MPhi with the phoP constitutive mutant strain CS022, which does not induce apoptosis in infected MPhi, does not result in presentation of a bacteria-derived antigen by bystander DCs unless the infected MPhi are induced to undergo apoptosis by treatment with lipopolysaccharide and ATP. DCs appear to be unique in their ability to present antigens derived from MPhi induced to undergo apoptosis by Salmonella, as bystander MPhi are not capable of presenting the bacteria-derived antigen despite the fact that they efficiently internalize the apoptotic cells. These data suggest that apoptosis induction by bacterial infection of MPhi may not be a quiescent death that allows the bacteria to escape recognition by the immune system, but rather may contribute to an antimicrobial immune response upon engulfment by bystander DCs.

Allelic imbalance and microsatellite instability in prostatic adenocarcinoma.

Although prostate cancer is one of the most common malignancies of males in Western countries, relatively little is known about the molecular mechanisms involved in tumor initiation and progression. Allelic loss studies have suggested the involvement of multiple tumor suppressor genes (TSGs), but few detailed studies of all chromosomes have been performed. In an effort to localize and identify candidate TSGs, we performed allelic imbalance (AI) studies on 55 prostate cancers, using 135 polymorphic microsatellite markers. For the entire chromosome. AI ranged from a low of 0% on chromosomes 14 and 20 to a high of 71% on chromosome 8. Chromosomal regions demonstrating at least twice the background frequency of AI (ranging from 20 to 69%) included 5q, 6q, 7q, 8p, 13, l6q, l8q, and 21. In addition, AI was examined for association with a number of clinicopathological parameters. AI on chromosomes 7 and 16 were each associated with greater age at diagnosis (P = 0.009 and 0.001, respectively), and AI on chromosomes 10, 16, and 18 was associated with aneuploidy/tetraploidy (P = 0.037, 0.013, and 0.054, respectively). Furthermore, AI on chromosome 5 was associated with a higher pathological stage (P = 0.021) and on chromosome 8 and 16 with a higher Gleason score (P = 0.027 and 0.041, respectively). No tumor exhibited a phenotype of widespread microsatellite instability. These results indicate that there likely exist multiple sites harboring candidate TSG in prostate cancer, some of which may have important clinical implications, and which argue against widespread microsatellite instability.

Macrophage and dendritic cell subsets in IBD: ALDH+ cells are reduced in colon tissue of patients with ulcerative colitis regardless of inflammation.

Disruption of the homeostatic balance of intestinal dendritic cells (DCs) and macrophages (MQs) may contribute to inflammatory bowel disease. We characterized DC and MQ populations, including their ability to produce retinoic acid, in clinical material encompassing Crohn's ileitis, Crohn's colitis and ulcerative colitis (UC) as well as mesenteric lymph nodes (MLNs) draining these sites. Increased CD14(+)DR(int) MQs characterized inflamed intestinal mucosa while total CD141(+) or CD1c(+) DCs numbers were unchanged. However, CD103(+) DCs, including CD141(+)CD103(+) and CD1c(+)CD103(+) DCs, were reduced in inflamed intestine. In MLNs, two CD14(-) DC populations were identified: CD11c(int)HLADR(hi) and CD11c(hi)HLADR(int) cells. A marked increase of CD11c(hi)HLADR(int) DC, particularly DR(int)CD1c(+) DCs, characterized MLNs draining inflamed intestine. The fraction of DC and MQ populations expressing aldehyde dehydrogenase (ALDH) activity, reflecting retinoic acid synthesis, in UC colon, both in active disease and remission, were reduced compared to controls and inflamed Crohn's colon. In contrast, no difference in the frequency of ALDH(+) cells among blood precursors was detected between UC patients and non-inflamed controls. This suggests that ALDH activity in myeloid cells in the colon of UC patients, regardless of whether the disease is active or in remission, is influenced by the intestinal environment.

Isolation of a cDNA encoding a novel zinc-finger protein from neuroblastoma x glioma NG108-15 cells.

A subtraction cDNA library was constructed from control hybrid NG108-15 (mouse neuroblastoma x rat glioma) cells and NG108-15 cells which had been treated for 48 h with the delta-opioid agonist D-Ala2-D-Leu5 enkephalin (DADLE) to down-regulate the delta-opioid receptor on these cells. Among the clones isolated from this library was NGD16-4, a 2768-bp clone encoding a putative 64-kDa protein containing 14 tandemly repeated zinc fingers (Zf) with high homology to the Krüppel family of Zf proteins. NGD16-4 also contains a region homologous to the A element of the Krüppel Associated Box (KRAB) domain, a domain recently linked to transcriptional repression. Southern and Northern analyses indicate that NGD16-4 is derived from the mouse genome. Northern analysis also demonstrates that expression of NGD16-4 mRNA is much higher in several mouse neuroblastoma cell lines than in mouse brain or other tissues. Although the function of NGD16-4 is unclear, the expression pattern of NGD16-4 indicates a possible association with the processes of differentiation or transformation in the mouse.

Antigen-presenting cells and anti-Salmonella immunity.

The present article summarizes studies aimed at addressing the role of antigen-presenting cell populations, particularly dendritic cells (DC), in the immune response to Salmonella typhimurium. Data from in vitro studies shed light on presentation of antigens expressed in Salmonella on major histocompatibility complex class I and class II molecules by infected DC and macrophages, and the activation state of DC following infection. Finally, data from in vivo studies addressing the role of DC and defined DC subsets during the host response to Salmonella using a murine infection model are discussed.

Analysis of a very large trinucleotide repeat in a patient with juvenile Huntington's disease.

A patient with juvenile Huntington's disease (HD) of probable maternal inheritance is reported. The expanded IT-15 allele was only detected with the use of modified PCR and Southern transfer techniques, which showed a CAG trinucleotide repeat expansion of approximately 250 repeats-the largest CAG expansion reported within the huntingtin gene. This case emphasizes the need for communication between the diagnostic laboratory and the clinician to define the molecular genetics of unusual cases.

Differential expression of opioid receptor genes in human lymphoid cell lines and peripheral blood lymphocytes.

The existence of receptors for opioid compounds on cells of the immune system has long been hypothesized, but has been very difficult to demonstrate unequivocally. We have used reverse-transcription polymerase chain reaction to obtain cDNA clones from the human MOLT-4 and CEM-3 T-leukemic cell lines which are nearly identical to portions of the delta and kappa opioid receptor cDNAs recently isolated from human brain and placenta, respectively. Northern analyses with riboprobes derived from the delta and kappa opioid receptor clones indicate these sequences are expressed at low levels in human peripheral blood lymphocytes and in several human lymphoid cell lines. Sequences corresponding to the mu opioid receptor cDNA were not detected in this study. The results suggest that delta and kappa opioid receptors may be responsible for mediating some direct effects of opioids in immune cells.

Enhancement of glycine receptor function by ethanol: role of phosphorylation.

1. The effects of several kinase inhibitors (staurosporine, GF 109203X, H89, KN62, genistein) and of the phosphatase inhibitor calyculin A were studied on the ethanol potentiation and on the function of homomeric alpha1 glycine receptor expressed in Xenopus oocytes using a two electrode voltage clamp recording technique. 2. The function of the homomeric alpha1 glycine receptor was not modified in Xenopus oocytes pretreated with kinase inhibitors or with the phosphatase inhibitor calyculin A. 3. The potentiation of the glycine receptor function induced by ethanol (10-200 mM) was significantly reduced in Xenopus oocytes pretreated with the PKC inhibitors staurosporine or GF 109203X. 4. No differences in propofol (2.5 microM) or halothane (250 microM) actions were found after exposure of Xenopus oocytes to staurosporine. 5. No differences in ethanol sensitivity were found after exposure of Xenopus oocytes expressing glycine alpha1 receptors to H89, KN62, genistein or to the phosphatase inhibitor calyculin A. 6. The mutant alpha1 (S391A), in which the PKC phosphorylation site at serine 391 was mutated to alanine, was less sensitive to the effects of ethanol than was the alpha1 wild type receptor. Moreover, the ethanol potentiation of the glycine receptor function was not affected by treatment with staurosporine in oocytes expressing alpha1 (S391A). 7. The splice variant of the alpha1 glycine receptor subunit, alpha1ins, containing eight additional amino acids and a potential phosphorylation site for PKA, did not differ from wild type for sensitivity to ethanol. 8. These results indicate that phosphorylation by PKC of the homomeric alpha1 glycine receptor subunit modulates ethanol potentiation, but not the function of the glycine receptor.

Subunit mutations affect ethanol actions on GABA(A) receptors expressed in Xenopus oocytes.

1. Mutations of specific amino acids were introduced in transmembrane domains (TM) of GABA(A) receptor alpha2, beta1 and gamma2L subunits. The effects of these mutations on the action of ethanol were studied using the Xenopus oocyte expression system and two-electrode voltage-clamp recording techniques. 2. Mutant alpha2 subunits containing S270I (TM2) or A291W (TM3) made the receptor more sensitive to GABA, as compared to wild-type alpha2beta1gamma2L receptor. The mutation S265I (TM2) of beta1 and S280I (TM2) or S30IW (TM3) in gamma2L subunits did not alter apparent affinity of the receptor for GABA. M286W (TM3) in the beta1 subunit resulted in a receptor that was tonically open. 3. Using an EC5 concentration of GABA, the function of the wild-type receptor with alpha2beta1gamma2L subunits was potentiated by ethanol (50-200 mM). The mutations in TM2 or TM3 of the alpha2 subunit diminished the potentiation by ethanol. The action of ethanol was also eliminated with a mutation in the TM2 site of the beta1 subunit. Ethanol produced significant inhibition of GABA responses in receptors containing the combination of alpha2 and beta1 TM2 mutants with a wild-type gamma2L subunit. A small but significant reduction in the potentiation by ethanol was observed with gamma2L TM2 and/or TM3 mutants. 4. From these results, we suggest that in heteromeric GABA(A) receptors composed of the alpha, beta and gamma subunits, ethanol may bind in a cavity formed by TM2 and TM3, and that binding to the alpha or beta subunit may be more critical than the gamma subunit.

Inactivation of hamster monomorphic N-acetyltransferase by vinyl fluorenyl ketone.

Arylamine N-acetyltransferases (NATs) are cytosolic enzymes that play important roles in the detoxification and activation of xenobiotic arylamines and their metabolites. Vinyl fluorenyl ketone (VFK) is a selective and potent active site-directed irreversible inhibitor of rat liver monomorphic NAT. The present study demonstrated that VFK is an active site-directed affinity label for hamster liver monomorphic NAT, but is a much less effective inactivator of the polymorphic N-acetyltransferase isozyme. The potency, irreversibility and selectivity of VFK make it a potentially valuable tool for characterization of NATs that exhibit acetyl donor specificity similar to that of hamster monomorphic NAT.

Bioactivation of N-arylhydroxamic acids by rat hepatic N-acetyltransferase. Detection of multiple enzyme forms by mechanism-based inactivation.

Enzymatic N,O-acyltransfer of carcinogenic N-arylhydroxamic acids such as N-hydroxy-2-acetylaminofluorene (N-OH-AAF) results in the production of reactive electrophiles that can bond covalently with nucleophiles and also can cause inactivation of acyltransferase activity in a mechanism-based manner. Incubation of partially purified rat hepatic N-acetyltransferases (NAT) with N-OH-AAF resulted in extensive inactivation of N-OH-AAF/4-aminoazobenzene (AAB) N,N-acetyltransferase and acetyl coenzyme A (AcCoA)/procainamide (PA) N-acetyltransferase activities, whereas AcCoA/p-aminobenzoic acid (PABA) N-acetyltransferase activity was inhibited only slightly. Affinity chromatography with Sepharose 6B 2-aminofluorene (2-AF) resulted in the separation of two NAT activities. NAT I primarily catalyzed the AcCoA-dependent acetylation of PABA; NAT II catalyzed, N,N-acetyltransfer (N-OH-AAF/AAB), AcCoA/PA N-acetyltransfer and N-OH-AAF N,O-acyltransfer (AHAT) activities. Most of the AcCoA/2-AF N-acetyltransferase activity eluted in the NAT II fraction. Results of inactivation experiments with N-OH-AAF and the NAT II fractions suggested that one NAT isozyme was responsible for catalyzing the N-OH-AAF/AAB, AcCoA/PA and N,O-acyltransfer reactions and that inactivation of NAT II correlated with the extent of covalent binding to protein. Further purification of the NAT II fractions by chromatofocusing resulted in a 1300-fold purification of the N-OH-AAF/AAB activity and the coelution of N-OH-AAF/AAB, AcCoA/PA and N,O-acyltransferase activities. These studies indicate that N,O-acyltransfer, arylhydroxamic acid-dependent N-acetylation of arylamines (N,N-acetyltransfer), and AcCoA-dependent N-acetylation of PA may be catalyzed by a common enzyme in rat liver, whereas a second enzyme is responsible for the AcCoA-dependent N-acetylation of PABA.

An isozyme-selective affinity label for rat hepatic acetyltransferases.

Affinity chromatography of an ammonium sulfate precipitate obtained from rat hepatic cytosol resulted in the separation of two fractions of N-acetyltransferase (NAT) activity. NATI catalyzed the S-acetylcoenzyme A (AcCoA)-dependent acetylation of p-aminobenzoic acid (PABA); NAT II catalyzed the N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-dependent acetylation of 4-amino-azobenzene (AAB) (N,N-acetyltransferase), the AcCoA-dependent acetylation of procainamide (PA), and the N-arylhydroxamic acid N,O-acyltransferase (AHAT) activity that results in the conversion of N-OH-AAF and related hydroxamic acids to electrophilic reactants. 1-(Fluoren-2-yl)-2-propen-1-one (vinyl fluorenyl ketone, VFK) was shown to be a potent and irreversible inactivator of NAT II activities. A 200-fold higher concentration of VFK was required to inactivate NAT I activity than was required for inactivation of NAT II activities. Similar selectivity in the inactivation of the isozymes was observed when experiments were conducted with enzyme preparations that contained both NAT I and NAT II activities. The presence of substrates and products of the NAT II-catalyzed reactions such as AcCoA, 2-acetylaminofluorene (2-AAF), and N-acetyl-4-aminoazobenzene (N-Ac-AAB) protected NAT II from the inactivating effects of VFK, providing evidence that VFK is an active site directed inhibitor (affinity label) of NAT II. Studies with 1-(fluoren-2-yl)-2-propan-1-one (EFK), an analogue of VFK in which the alpha, beta-unsaturated vinyl ketone group of VFK has been replaced with an ethyl ketone group, demonstrated that the conjugated ketone of VFK is required for inactivation of enzyme activity. The results of these studies suggest that agents such as VFK should have utility as probes of acetyltransferase multiplicity and in the investigation of the active site topography of the enzymes.

Irreversible inhibition of rat hepatic transacetylase activity by N-arylhydroxamic acids.

Both N-hydroxy-2-acetamidofluorene (N-OH-AAF) and the heterocyclic analogue, 2-(N-hydroxyacetamido)carbazole (N-OH-AAC), were shown to be mechanism-based irreversible inhibitors (suicide inhibitors) of partially purified rat hepatic N-acetyltransferase (NAT) activity. Although N-OH-AAC exhibited an apparent first-order inactivation rate constant (ki) that was 7-fold lower than that of N-OH-AAF, their relative ki/KD values indicate that N-OH-AAC was the more potent and efficient inactivator of transacetylase activity. Inactivation of NAT activity by these N-arylhydroxamic acids appeared to involve contributions by electrophiles that react with the enzyme subsequent to release from the active site and by electrophiles that remain complexed with the active site. The irreversible nature of the enzyme inactivation was demonstrated by the failure to recover transacetylase activity upon either extensive dialysis or gel filtration of preparations that had been subjected to incubation with N-OH-AAF or N-OH-AAC. The use of the nucleophile N-acetylmethionine to trap the electrophilic reactants formed in the transacetylase-catalyzed bioactivation process resulted in a lower rate and extent of formation of methylthio adducts with N-OH-AAC than with N-OH-AAF. The results of this study indicate that N-OH-AAF and N-OH-AAC have potential for use as tools in the investigation of rat hepatic transacetylases.

Expression of OBCAM-related cDNA clones in Cos 1 cells: evidence for a phosphatidylinositol linkage to the cell membrane.

Previously, our laboratory purified and isolated the cDNA for OBCAM (opioid binding cell adhesion molecule) from bovine brain, as well as highly homologous rat brain cDNA clones, SG13 and DUZ-1. Structural similarities with members of the immunoglobulin superfamily suggest a possible role for OBCAM in cell adhesion and recognition, while studies in our own laboratory suggest that OBCAM is important in the regulation of opioid binding and signal transduction. However, OBCAM lacks a putative transmembrane domain, and its possible mode of linkage to the cellular membrane has not been studied. Upon transfection of Cos 1 cells with SG13 and DUZ-1 cDNAs, the OBCAM-homologous proteins were expressed on the surface of the Cos 1 cells. These proteins were released from the membrane of the Cos 1 cells upon digestion with phosphatidylinositol-specific phospholipase C (PI-PLC), demonstrating that they are linked to the membrane via a phosphatidylinositol (PI) linkage. These results are consistent with a role for OBCAM in cell recognition and adhesion, as well as in cellular signaling.

Molecular cloning of a novel protein regulated by opioid treatment of NG108-15 cells.

A new cDNA clone, NGD5, has been identified from a subtraction cDNA library constructed with mRNA isolated from control neuroblastoma x glioma NG108-15 cells and cells treated for 48 h with the delta-opioid agonist, D-Ala2, D-Leu5 enkephalin (DADLE). NGD5 mRNA is decreased, in a naloxone-reversible manner, upon long-term treatment of NG108-15 cells with DADLE, indicating that this clone may be related to opioid receptor function. Northern analysis indicates that NGD5 mRNA is expressed in rat brain. Two similar nearly full-length NGD5 clones, NGD5A and NGD5B, were isolated from a lambda gt10 NG108-15 cDNA library and sequenced. The predicted 40-kDa peptide encoded for by the NGD5 cDNA has no significant homology to the recently cloned mu, delta or kappa opioid receptors nor to any other known proteins.

Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned mu, delta, and kappa opioid receptors.

A rat brain cDNA library was screened for clones homologous to the recently cloned mouse delta-opioid receptor (DOR-1). Among the clones isolated was Hyp 8-1, a clone with a unique nucleotide sequence capable of encoding a putative protein which is 57-58% identical to the amino acid sequences of the cloned delta, mu and kappa opioid receptors, indicating a close relationship of Hyp 8-1 with the opioid receptor family. Several cDNAs representing possible splice variants of Hyp 8-1 were also isolated. Binding studies of COS-7 cells transfected with clone Hyp 8-1 failed to demonstrate specific binding with several 3H-opioid ligands. In situ hybridization studies indicate that the mRNA for Hyp 8-1 is distributed discretely throughout the rat brain, in an overall pattern which is different from that of several other G-protein-coupled seven transmembrane receptors. Thus, it is likely that the Hyp 8-1 cDNA encodes a novel peptide receptor.

Expression of alternate forms of brain opioid 'orphan' receptor mRNA in activated human peripheral blood lymphocytes and lymphocytic cell lines.

We screened a PHA (phytohemagglutinin)-activated human lymphocyte cDNA library for clones with homology to the recently cloned brain opioid receptors. A cDNA clone, AT7-5EU, was isolated which encodes the opioid 'orphan' receptor, a molecule with very high homology to the opioid receptor gene family, but which has not been shown to bind opioids or any other known compounds. The protein coding region of AT7-5EU has complete homology with a reported opioid 'orphan' clone isolated from human brain, but the 5' untranslated regions of AT7-5EU and the human brain clones are divergent, suggesting mechanisms for tissue-specific expression of this receptor. Northern analysis of AT7-5EU mRNA demonstrates the expression of this message in human lymphocytic cell lines of both B-and T-cell lineages. Furthermore, analysis of mRNA from human peripheral blood lymphocytes demonstrates that activation of the lymphocytes with PHA results in at least a 10-fold induction of the AT7-5EU message. These results suggest that the opioid 'orphan' receptor may have an important immunological function in addition to its function in the nervous system.

Stable expression of human glycine alpha1 and alpha2 homomeric receptors in mouse L(tk-) cells.

We report the development of two mouse fibroblast-like stably-transfected cell lines (alpha1-62-4 and alpha2-B36-1) that express human alpha1 or alpha2 glycine receptor subunits, respectively. Transfected cDNAs were cloned into the pMSGneo expression vector, for which transcription is controlled by the dexamethasone-inducible MMTV promoter. Patch-clamp electrophysiological recordings revealed that the alpha1 or alpha2 glycine receptor subunits expressed in these cells form functional glycine receptors that are inhibited by strychnine and picrotoxin. Glycine activated currents in these cells with EC50s of 101+/-7 or 112+/-23 microM for cells stably expressing alpha1 or alpha2 receptors, respectively. As indicated by assays of glycine-stimulated 36Cl-- uptake, these cells express glycine receptors only after treatment with dexamethasone. In order to measure expression of the glycine alpha1 or alpha2 receptor protein, we produced a new anti-alpha1/alpha2 glycine receptor antibody (anti-alpha GR). Western blot analysis with this antibody showed a band of approximately 48 kDa only in homogenates from cells which had been transfected with the glycine alpha1 or alpha2 receptor cDNAs. Thus, through use of this stable expression system, we successfully produced cell lines expressing strychnine-sensitive glycine receptors that display similar functional characteristics to homomeric glycine receptors expressed in other systems. These stably transfected cells should provide a useful in vitro system for the study of the physiology and pharmacology of strychnine-sensitive glycine receptors.

The influence of curli, a MHC-I-binding bacterial surface structure, on macrophage-T cell interactions.

Escherichia coli express thin surface fimbriae called curli which bind soluble matrix proteins and major histocompatibility complex (MHC)-I molecules. The present study addressed the ability of purified curli or curliated E. coli to influence peptide presentation on MHC-I, T cell proliferation and bacterial uptake by macrophages. In vitro studies with curli-proficient E. coli YMel and the isogenic curli-deficient strain YMel-1, both expressing the model antigen Crl-OVA, showed that curli expression by E. coli does not appear to influence the efficiency by which the bacteria are processed by murine macrophages for OVA(257-264) presentation on K(b). Furthermore, curli expression by E. coli did not influence the binding of exogenously added OVA(257-264) peptide to K(b) on the surface of prefixed macrophages. In addition, neither curliated nor non-curliated heat-killed bacteria influenced proliferation of either murine or human T cells stimulated with anti-CD3. Finally, curliated E. coli adhered to and were internalized by macrophages from C57BL/6 and MHC-I-deficient TAP1(-/-) mice equally well. Together these studies show that curli expression by E. coli does not appear to influence phagocytic processing of bacteria expressing Crl-OVA for OVA(257-264)/K(b) presentation, the binding of exogenously added OVA(257-264) to K(b) or T cell proliferation. In addition, although curli expression by E. coli enhances bacterial interaction with macrophages, curli interaction with MHC-I does not significantly contribute to this adherence.