Types Of Chimeras Research Articles

TLR2 Stimulation of Intrinsic Renal Cells in the Induction of Immune-Mediated Glomerulonephritis

Infection may exacerbate organ-specific autoimmune disease such as glomerulonephritis. This may occur in the absence of a measurable effect on the adaptive immune response, and the mechanisms responsible are not fully understood. To investigate this, we have studied the effect of TLR2 ligation by the synthetic ligand Pam(3)CysSK(4) on the development of glomerulonephritis in mice. We demonstrated that glomerular inflammation induced by passive administration of nephrotoxic Ab does not occur in the absence of TLR2 stimulation, with a strong synergy when Ab deposition and TLR2 stimulation occur together. Parameters of glomerular inflammation were neutrophil influx, thrombosis, and albuminuria. To investigate the relative contribution of TLR2 on bone marrow-derived cells and intrinsic renal cells, we constructed bone marrow chimeras. Nephrotoxic Ab and TLR2 ligation caused a neutrophil influx in both types of chimera above [corrected] that seen in sham chimeras totally TLR2 deficient [corrected] Albuminuria was seen in both types of chimera above that seen in sham chimeras that were totally TLR2 deficient. This was greater in chimeras with TLR2 present on bone marrow-derived cells. To find a potential mechanism by which intrinsic renal cells may contribute toward disease exacerbation, mesangial cells were studied and shown to express TLR2 and MyD88. Wild-type but not TLR2-deficient mesangial cells produced CXC chemokines in response to stimulation with Pam(3)CysSK(4). These results demonstrate that TLR2 stimulation on both bone marrow-derived and resident tissue cells plays a role in amplifying the inflammatory effects of Ab deposition in the glomerulus.

Combined Free Partial Vastus Lateralis with Anterolateral Thigh Perforator Flap for Reconstruction of Extensive Composite Defects

007-1226/$ see front matter q 20 i:10.1016/j.bjps.2005.04.022 * Corresponding author. Tel.: C31 1 E-mail address: sophofer@hotmail. Summary Myocutaneous (MC) free flaps are useful for many reconstructive indications. Perforator flaps have become standard of care. The anterolateral thigh flap (ALT) donor site is popular. With the ALT flap varying sizes of vastus lateralis (VL) muscle can be harvested as a MC flap. The skin islands of these flaps have a great range of freedom when dissected on their perforator. It was hypothesised that the VL–ALT perforator flap would offer adequate tissue volume combining maximal freedom in planningwithminimal donor sitemorbidity. FromNovember 2001 to February 2003 a free partial VL with ALT perforator flap was used in 11 patients to reconstruct large defects. Indications for adding a muscular component were exposed bone, skull base, (artificial) dura, or osteosynthesis material, open sinuses, and lack of muscular bulk. Flaps were planned as standard ALT flaps, after which three types of dissection were performed: I. trueMCflap; II.muscleflapwitha skin islandononeperforator,whichcouldbe rotatedup to1808; III. chimera skin perforatorflapwithmuscle being harvestedona separatebranch from the source vessel or on a side branch of the skin perforator. Mean skin size of theMCALT flaps was 131 cm. Meanmuscle part size of the MC-ALT flaps was 268 cm. Muscular parts were custom designed for all defects. No total or partial flap failures were seen. Colourmismatchwas seen in6of8patients,whenskinwasused in the facial area in this all white population. Excessive flap bulk was found in 8 of 11 patients at 6 weeks, however, only in 2of 11patients after 6months. Patientswere satisfiedwith the functional result (8 of 11 patients) as well as the cosmetic result of their reconstruction (7 of 11 patients). All less satisfiedpatientshad receivedtheirflap forexternal facial skin reconstruction.Donor sitemorbiditywasminimal. The combined free partial VLwithALTperforator flapproved valuable as a (chimera type) MC flap with maximal freedom of planning to meet specific reconstructive demands and minimal donor site morbidity. q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved. British Journal of Plastic Surgery (2005) 58, 1095–1103

Expression and Modulation of Carbohydrate-Binding Protein Galectin-3 in Multiple Myeloma Cells by Combined Treatment with GCS-100 and Dexamethasone.

Galectins belong to the family of mammalian lectins sharing an evolutionary conserved sequence in their carbohydrate recognition binding (CRD) domains and exhibit a characteristic affinity for beta-galactoside-sugars. Based on their structure and CRD, galectins have been categorized into three different categories: 1) Prototype (Gal-1, 2, 5, 7, 10, 11, 13, and 14), 2) Chimera type (Gal-3), and 3) Tandem repeat type (Gal-6, 8, 9, and 12). In particular, Galcetin-3 (Gal-3) is associated with growth, differentiation, adhesion, RNA processing, and apoptosis ( Apoptosis , 2005, 10: 267–75). Previous studies show that Gal-3 is expressed on tumor cell surface and mediate the transformation and metastasis of tumor cells in vivo ( Apoptosis , 2005, 10: 267–75). Here, we show that Gal-3 is differentially expressed in multiple myeloma (MM) cell lines and purified patient cells, as determined by both western blotting and polymerase chain reaction (PCR). Importantly, Gal-3 shares structural similarities with the anti-apoptotic protein Bcl-2: both proteins are rich in proline, glycine, and alanine amino acid residues in their NH-2-terminal domains and contain anti-death motif Asp-Trp-Gly-Arg (NWGR) in their C-terminal domains ( Proc Natl Acad Sci USA , 1996, 93: 6737–42). Gal-3, like Bcl-2, is therefore a potential therapeutic target. In this context, modified citrus pectins (MCP) have been reported to bind and inhibit Gal-3-induced human umbilical endothelial cell migration and micro vessel tube formation ( Am J Pathol. 2000, 156:899–909) as well as block tumor growth and metastasis (J Natl Cancer Inst. 2002, 94:1854–62). GCS-100 is a MCP currently under clinical development. Here we show that GCS-100 binds to human recombinant Gal-3. In MM cells, GCS-100 triggers apoptosis and enhances anti-tumor activity of the conventional agent Dexamethasone. We examined whether GCS-100-, Dex- or GCS-100 + Dex-induced MM cell death modulates Gal-3 or Bcl-2 expression. Treatment of MM.1S cells with either GCS-100 or Dex alone does not alter Gal-3 or Bcl-2 expression. However, combined treatment of MM cells with sub-toxic concentrations of GCS-100 and Dex markedly attenuates Gal-3 expression, without any changes in Bcl-2 levels, suggesting that alterations in Gal-3 are specific. Previous studies showed that Gal-3 inhibits mitochondrial pro-apoptotic protein cytochrome-c (cyto-c), and our results show that GCS-100 + Dex triggers the release of cyto-c from mitochondria to cytosol. Together, these data suggest that the combination of GCS-100 + Dex overcomes the inhibitory effect of Gal-3 on cyto-c, thereby inducing apoptosis. Since Gal-3 is widely expressed in MM cells, its inhibition may augment the anti-tumor activity of therapeutic regimens.

The combined free partial vastus lateralis with anterolateral thigh perforator flap reconstruction of extensive composite defects

Myocutaneous (MC) free flaps are useful for many reconstructive indications. Perforator flaps have become standard of care. The anterolateral thigh flap (ALT) donor site is popular. With the ALT flap varying sizes of vastus lateralis (VL) muscle can be harvested as a MC flap. The skin islands of these flaps have a great range of freedom when dissected on their perforator. It was hypothesised that the VL-ALT perforator flap would offer adequate tissue volume combining maximal freedom in planning with minimal donor site morbidity. From November 2001 to February 2003 a free partial VL with ALT perforator flap was used in 11 patients to reconstruct large defects. Indications for adding a muscular component were exposed bone, skull base, (artificial) dura, or osteosynthesis material, open sinuses, and lack of muscular bulk. Flaps were planned as standard ALT flaps, after which three types of dissection were performed: I. true MC flap; II. muscle flap with a skin island on one perforator, which could be rotated up to 180 degrees ; III. chimera skin perforator flap with muscle being harvested on a separate branch from the source vessel or on a side branch of the skin perforator. Mean skin size of the MC-ALT flaps was 131 cm2. Mean muscle part size of the MC-ALT flaps was 268 cm3. Muscular parts were custom designed for all defects. No total or partial flap failures were seen. Colour mismatch was seen in 6 of 8 patients, when skin was used in the facial area in this all white population. Excessive flap bulk was found in 8 of 11 patients at 6 weeks, however, only in 2 of 11 patients after 6 months. Patients were satisfied with the functional result (8 of 11 patients) as well as the cosmetic result of their reconstruction (7 of 11 patients). All less satisfied patients had received their flap for external facial skin reconstruction. Donor site morbidity was minimal. The combined free partial VL with ALT perforator flap proved valuable as a (chimera type) MC flap with maximal freedom of planning to meet specific reconstructive demands and minimal donor site morbidity.

The virion-associated Gag–Pol is decreased in chimeric Moloney murine leukemia viruses in which the readthrough region is replaced by the frameshift region of the human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) requires a programmed −1 translational frameshift event to synthesize the precursor of its enzymes, Gag–Pol, when ribosomes from the infected cells translate the full-length viral messenger RNA. Translation of the same RNA according to conventional translational rules produces Gag, the precursor of the structural proteins of the virus. The efficiency of the frameshift controls the ratio of Gag–Pol to Gag, which is critical for viral infectivity. The Moloney murine leukemia virus (MoMuLV) uses a different strategy, the programmed readthrough of a stop codon, to synthesize Gag–Pol. In this study, we investigated whether different forms of the HIV-1 frameshift region can functionally replace the readthrough signal in MoMuLV. Chimeric proviral DNAs were obtained by inserting into the MoMuLV genome the HIV-1 frameshift region encompassing the slippery sequence where the frameshift occurs, followed by the frameshift stimulatory signal. The inserted signal was either a simple stem-loop, previously considered as the stimulatory signal, or a longer bulged helix, now shown to be the complete stimulatory signal, or a mutated version of the complete signal with a three-nucleotide deletion. Although the three chimeric viruses can propagate essentially as the wild-type virus in NIH 3T3 cells, single-round infectivity assays revealed that the infectivity of the chimeric virions is about three to fivefold lower than that of the wild-type virions, depending upon the nature of the frameshift signal. It was also observed that the Gag–Pol to Gag ratio was decreased about two to threefold in chimeric virions. Comparison of the readthrough efficiency of MoMuLV to the HIV-1 frameshift efficiency, by monitoring the expression of a luciferase reporter in cultured cells, revealed that the frameshift efficiencies were only 30–60% of the readthrough efficiency. Altogether, these observations indicate that replacement of the readthrough region of MoMuLV with the frameshift region of HIV-1 results in virions that are replication competent, although less infectious than wild-type MoMuLV. This type of chimera could provide an interesting tool for in vivo studies of novel drugs targeted against the HIV-1 frameshift event.

Galectin-3 Precipitates as a Pentamer with Synthetic Multivalent Carbohydrates and Forms Heterogeneous Cross-linked Complexes

Galectin-3 is unique among the galectin family of animal lectins in its biological activities and structure. Most members of the galectin family including galectin-1 possess apoptotic activities, whereas galectin-3 possesses anti-apoptotic activity. Galectin-3 is also the only chimera type galectin and consists of a nonlectin N-terminal domain and a C-terminal carbohydrate-binding domain. Recent sedimentation equilibrium and velocity studies show that murine galectin-3 is a monomer in the absence and presence of LacNAc, a monovalent sugar. However, quantitative precipitation studies in the present report indicate that galectin-3 precipitates as a pentamer with a series of divalent pentasaccharides with terminal LacNAc residues. Furthermore, the kinetics of precipitation are fast, on the order of seconds. This indicates that although the majority of galectin-3 in solution is a monomer, a rapid equilibrium exists between the monomer and a small percentage of pentamer. The latter, in turn, precipitates with the divalent oligosaccharides, resulting in rapid conversion of monomer to pentamer by mass action equilibria. Mixed quantitative precipitation experiments and electron microscopy suggest that galectin-3 forms heterogenous, disorganized cross-linking complexes with the multivalent carbohydrates. This contrasts with galectin-1 and many plant lectins that form homogeneous, organized cross-linked complexes. The results are discussed in terms of the biological properties of galectin-3.

Pathogenesis of hippocampal neuronal death after hypoxia–ischemia changes during brain development

Transient hypoxia–ischemia (HI) leads to delayed neuronal death in both mature and immature neurons but the underlying mechanisms are not fully understood. To understand whether the pathogenesis of HI-induced neuronal death is different between mature and immature neurons, we used a rat HI model at postnatal days 7 (P7), 15 (P15), 26 (P26) and 60 (P60) in order to investigate ultrastructural changes and active caspase-3 distribution in HI-injured neurons as a function of developmental age. In P7 pups, despite more than 95% of HI-injured neurons highly expressing active caspase-3, most of these active caspase-3-positive neurons revealed mixed features of apoptosis and necrosis (a chimera type) under electron microscopy (EM). Classical apoptosis was observed only in small populations of HI-injured P7 neurons. Furthermore, in rats older than P7, most HI-injured neurons displayed features of necrotic cell death under EM and, concomitantly, active caspase-3-positive neurons after HI declined dramatically. Classical apoptosis after HI was rarely found in neurons older than P15. In P60 rats, virtually all HI-injured neurons showed the shrinkage necrotic morphology under EM and were negative for active caspase-3. These results strongly suggest that pathogenesis of HI-induced neuronal death is shifting from apoptosis to necrosis during brain development.

Normal B cell homeostasis requires B cell activation factor production by radiation-resistant cells.

The cellular source of B cell activation factor (BAFF) required for peripheral B cell survival/maturation is unknown. To determine the nature of BAFF-producing cells we established and analyzed reciprocal bone marrow (BM) chimeras with wild-type (WT) and BAFF-deficient mice. The results revealed that BAFF production by radiation-resistant stromal cells is completely sufficient to provide a necessary signal for B cell survival/maturation, as BAFF−/− BM cells transferred into lethally irradiated WT mice gave rise to normal numbers of follicular (FO) and marginal zone (MZ) B cell subpopulations. On the other hand, transfer of WT BM into BAFF−/−lethally irradiated mice resulted only in minimal reconstitution of mature FO B cells and no restoration of MZ B cells. Thus, in the absence of BAFF+/+stromal cells, BAFF production by BM-derived cells, presumably by macrophages, dendritic cells, and/or neutrophils, was not at all sufficient to support normal B cell homeostasis. Interestingly, immunization of both types of chimeras stimulated high levels of antigen-specific antibody secretion, indicating that either stromal cell– or hematopoietic cell–derived BAFF is sufficient for B cell antibody responses.

Characterization of the Xenopus Galectin Family: THREE STRUCTURALLY DIFFERENT TYPES AS IN MAMMALS AND REGULATED EXPRESSION DURING EMBRYOGENESIS

We have isolated six novel galectin cDNAs from a Xenopus laevis kidney cDNA library. The newly identified X. laevis galectins (xgalectins) comprise one proto type (xgalectin-Vb), one chimera type (xgalectin-VIIa), and four tandem repeat types (xgalectin-IIb, -IIIb, -VIa, and -VIIIa). Thus, together with those mentioned in our previous work (Shoji, H., Nishi, N., Hirashima, M., and Nakamura, T. (2002) Glycobiology 12, 163-172), the 12 xgalectins are classified into three types based on their domain structures, as in mammals. The xgalectins whose counterparts in other species have not been identified (xgalectin-IVa, -Vb, and -VIa) were confirmed to possess lactose-binding activity by expression of their recombinant forms. This shows that they truly function as animal lectins. The protein purification study revealed that the major xgalectins in kidney are xgalectin-Ib, -IIa, -IIb, -IIIa, and -VIIa. The mRNAs of xgalectin-IIb, -IIIb, -Vb, and -VIa were localized to specific adult tissues, whereas those of xgalectin-VIIa and -VIIIa were broadly distributed. The temporal expression patterns of the mRNAs of the 12 xgalectins during embryogenesis were analyzed and categorized into three groups: 1) mRNA observed to exist throughout embryogenesis, i.e. maternal mRNA also exists (xgalectin-Ia, -IIa, -IIIa, -IIIb, -Va, -VIIa, and -VIIIa); 2) mRNA observed from the gastrula stage (xgalectin-VIa); and 3) mRNA observed from the tail bud or the tadpole stage (xgalectin-Ib, -IIb, -IVa, and -Vb). The mRNA of the most abundant xgalectin in embryos, xgalectin-VIIa, was localized to the surface layer of embryos, the epidermis, the cement gland, and various placodes. Xgalectin-VIIa protein was also observed to exist throughout embryogenesis by Western blot analysis with specific antiserum. These results show that the expression of each member is spatiotemporally regulated from eggs to adulthood, suggesting that galectins play multiple roles not only in adults, but also in development.

T Lymphocytes of Allophenic Mice Do Not Reject Spleen Colony-Forming Units of the Parental Genotypes

Self-tolerance in BALB/c<-->C57BL/6 and BALB/c<-->B10.SM (SM) allophenic mice is examined in this study. Chimerism was determined by coat mosaicism, using electrophoresis of allozyme variants at the glucose phosphate isomerase-1 (GPI-1) in peripheral blood erythrocytes and a cytotoxity test for lymph node (LN) lymphocytes and blood nucleated cells. An age-dependent increase in the percent content of BALB/c erythrocytes was observed in both types of chimeras. The immunological status of chimeras was assessed by T cell interactions with spleen colony-forming cells (SFU-S) able to form colonies in the recipient spleens. LN lymphocytes of the chimeras, together with bone marrow cells (BMC) of the parental genotype, were transplanted to lethally irradiated F1 hybrids. Chimera T lymphocytes did not inactivate CFU-S of the parental genotype and colonies formed in the spleens of F1 hybrids. The absence of splenic colonies in (C x B6)F1 hybrids, in which BMC from B6 had been transplanted (either in combination with chimera lymphocytes or alone), was due to the reaction of host natural killer (NK) cells. The results obtained testify to the permanent immunological tolerance of chimera T lymphocytes to BMC antigens of the both parental genotypes. The chimeric shift in the erythrocyte population is not coupled to disturbances in self-tolerance of chimeric mice.

Silent Philadelphia Chromosome: A Distinct Developmental Stage in a Philadelphia Chromosome-Positive Chronic Myeloproliferation?

In this paper, a patient is described who presented with peripheral blood and bone marrow features uncharacteristic of chronic granulocytic leukemia, which proved to be Philadelphia (Ph) chromosome-positive by metaphase and interphase cytogenetic analyses but lacked the p210 type of BCR/ABL fusion gene mRNA product by two different sensitive RT-PCR assays. In the course of the 32-month follow-up with a termination into a myeloblastic crisis, molecular investigations were performed four times. They indicated a constantly high rate of Ph positive cells and lack of BCR/ABL mRNA expression, except in the second investigation, when the patient showed reverse transcription polymerase chain reaction positivity with b3/a2 type of chimera, fusion gene mRNA expression, and a striking change in the bone marrow histology. Our findings might indicate that the dormant Ph chromosome state may exist not only at the primitive progenitor, but also at the entire peripheral blood cell compartment level.

Fusions to members of fibroblast growth factor gene family to study nuclear translocation and nonclassic exocytosis

Publisher Summary The fibroblast growth factor (FGF) gene family includes at least 19 genes encoding proteins that regulate proliferation, differentiation, motility, and survival of cells of neuroectodermal and mesodermal origin. This chapter describes the fusions to members of FGF gene family to study nuclear translocation and nonclassic exocytosis. The two most abundantly expressed representatives of the family are the prototypes FGF-1 and FGF-2, which are involved in the regulation of a wide variety of developmental, pathological, and regenerative processes in mammalian organisms, including humans. Among these processes are mesodermal induction, limb formation, angiogenesis, skeletal muscle growth and regeneration, the growth and metastasis of certain types of solid tumors, atherosclerosis, and restenosis. The chapter discusses the types of protein chimeras used for the study of fibroblast growth. The protein fusion approach to the study of FGF translocations is not limited to the use of reporter genes. Known nuclear localization signal (NLS) sequences from other proteins have been used for fusion with the FGFs to direct their nuclear translocation. A comparative study of FGF-1 and FGF-2 nuclear localization and release includes the shuffling of homologous regions/domains between these two proteins.

Expression and activity of chimeric molecules between human UDP-galactose transporter and CMP-sialic acid transporter.

Human UDP-galactose transporter (hUGT1) and CMP-sialic acid transporter (hCST) are related Golgi proteins with eight putative transmembrane helices predicted by computer analysis. We constructed chimeric molecules in which segments of various lengths from the C- or N-terminus of hUGT1 were replaced by corresponding portions of hCST. The chimeras were transiently expressed in UGT-deficient mutant Lec8 cells, and their UGT activity was assessed by the binding of GS-II lectin to the transfected cells. The replacement of either the N- or C-terminal cytoplasmic segment by that of hCST did not affect the expression or activity of hUGT1. A chimera in which the eighth helix and the C-terminal tail were replaced also retained the UGT activity, indicating that this helix is not involved in the determination of substrate specificity. In contrast, three types of chimeras, in which the first helix, the first and the second helices, and a segment from the seventh helix to the C-terminus were replaced, respectively, were expressed very infrequently in the transfected cells, and had no UGT activity. They are likely folded incorrectly and degraded by a quality-control system, since the amounts of their mRNAs were normal and the proteins were mainly localized in the ER. The first and the seventh helices are important for the stability of the transporter protein.

Differential Control of Autoantibodies and Lymphoproliferation by Fas Ligand Expression on CD4+ and CD8+ T Cells In Vivo

We have previously shown that the gld autoimmune syndrome is suppressed in lethally irradiated gld mice reconstituted with a mixture of normal and gld bone marrow (BM). Furthermore, in vivo depletion of normal Thy-1+ cells restores lymphoproliferation and autoantibody production in such chimeras, suggesting that T cells bearing Fas ligand are responsible for correcting the gld defect. In this study, mixed-BM chimeras lacking either normal CD4+ (B6CD4KO-B6gld) or normal CD8+ T cells (B6CD8KO-B6gld) were generated to determine the contribution of the normal T cell subsets to disease suppression. Lymphoproliferation was completely suppressed in B6CD4KO-B6gld chimeras but only modestly in B6CD8KO-B6gld chimeras. On the other hand, both types of mixed-BM chimeras had incomplete effects on the suppression of serum autoantibodies when compared with B6gld reconstituted with isologous BM. These results suggest that both T cell subsets provide Fas ligand to suppress immune cells responsible for autoantibody production; however, CD8+ T cells are mainly responsible for preventing lymphoproliferation.

Extensive Mitochondrial Introgression from Pinus pumila to P. parviflora var. pentaphylla (Pinaceae)

species exhibit a paternal chloroplast inheritance and a maternal mitochondrial inheritance. The levels and patterns of cpDNA and mtDNA introgression between the two pine species, P. pumila and P. parviflora var. pentaphylla, were examined at three mountain sites in Japan. The pine species were examined by using PCR-based diagnostic genetic markers of cpDNA and mtDNA. The survey which was carried out in multiple hybrid zones demonstrated a generality in the uni-directional pattern of cytoplasmic gene flow between the two pine species, i.e. paternal cpDNA flowed from P. parviflora var. pentaphylla to P. pumila, and in contrast, maternal mtDNA flowed from P. pumila to P. parviflora var. pentaphylla. Whenever plants which had a non-native combination of cpDNA and mtDNA were observed, they always had the cpDNA haplotype of P. parviflora var. pentaphylla and the mtDNA haplotype of P. pumila. The existence of only this type of cytoplasmic chimera may suggest that F1 hybrids are successfully produced only in the crossing of P. pumila as the maternal parent and P. parviflora var. pentaphylla as the paternal parent. The present study also detected extensive mtDNA capture in populations of P. parviflora var. pentaphylla located in the southern and middle parts of the Ohu Mountains, Tohoku, Japan. In that area, nearly all of the plants examined had the mtDNA haplotype of P. pumila. The extensive mtDNA introgression suggests that seed flow could be an effective medium for interspecific gene exchange.

Antigen-presenting Cells in Experimental Autoimmune Uveoretinitis

The present study attempts to identify the antigen-presenting cells in the retina, utilizing bone marrow-transplanted chimeric rats. Two types of chimeras were used: one produced by transplanting bone marrow cells from F1hybrids of Lewis and Brown Norway (BN) into sublethally irradiated Brown Norway rats (LBN/F1→BN), followed by adoptive transfer of S-antigen-specific T cells obtained from Lewis rats; the second produced by transplanting bone marrow cells from BN rats into sublethally irradiated F1hybrids (BN→LBN/F1), followed by adoptive transfer of S-antigen-specific T cells obtained from F1hybrids. As controls, Lewis, F1hybrids and BN rats also received adoptive transfer of syngeneic uveitogenic T cell lines. All animals were killed on the seventh day after adoptive transfer and their eyes and pineal glands were analysed immunohistochemically, utilizing antibody directed against Lewis specific MHC class II molecules(OX-3). The analyses revealed the development of uveoretinitis and pinealitis in both types of chimeras and in the Lewis and F1hybrid rats. BN rats did not develop uveoretinitis. OX-3-positive cells were found in the retina and the pineal glands of both types of chimeras, and in the Lewis and F1hybrid rats but not in the BN rats. These cells in the retina expressed dendritic morphology and perivascular distribution. Retinal pigment epithelia, Müller cells and the vascular endothelia of both chimeras, the two strains, and the F1hybrid rats did not demonstrate OX-3-positive staining. These results suggest that the bone marrow-derived cells in the retina and pineal gland may present S-antigen to T cells, initiating the cascade of uveoretinitis and pinealitis.

Chimeras of the Native Form or Achondroplasia Mutant (G375C) of Human Fibroblast Growth Factor Receptor 3 Induce Ligand-Dependent Differentiation of PC12 Cells

Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to those of FGFR1, to which only positive effects have been ascribed, in PC12 cells. Therefore, its regulatory effects on bone growth likely result from cellular contexts and not the induction of a unique FGFR3 signaling pathway.

Selective Interaction of hsp90 with an Estrogen Receptor Ligand-binding Domain Containing a Point Mutation

The 90-kDa heat shock protein (hsp90) has been implicated in modulating steroid receptor function in vitro and in vivo. Previous studies have suggested that hsp90 interacts with large portions of the estrogen receptor (ER) ligand-binding domain and sequences of the receptor required for stable DNA binding. To characterize the interaction of the ER ligand-binding domain with hsp90, we have compared the properties of chimeras created by coupling the ligand-binding domain to the constitutive transactivator VP16-GAL. Two types of chimeras were created: VP16-GAL-ERG, containing the wild-type ligand-binding domain derived from the cDNA HEG0, and VP16-GAL-ERV, containing the substitution G400V derived from the ligand-binding domain of the original ER cDNA isolate, HE0. The G400V mutation alters the physical properties of VP16-GAL-ERV by rendering it hormone-dependent for DNA binding and more strongly dependent on estradiol for transactivation compared with VP16-GAL-ERG. Glycerol gradient analyses and chemical cross-linking/coimmunoprecipitation showed that, unlike VP16-GAL-ERG, VP16-GAL-ERV formed stable complexes with hsp90 in vitro. These data show that hsp90 selectively recognizes the altered ER ligand-binding domain containing the G400V substitution and indicate that the wild-type ER ligand-binding domain of VP16-GAL-ERG does not interact with hsp90 in vitro. Hormone binding studies showed that the ligand-binding domain of VP16-GAL-ERV was destabilized by incubation in the presence of high concentrations of salt or in the absence of sodium molybdate, conditions that disrupt its interaction with hsp90. The ligand-binding domain of the Val-400 ER thus behaves similarly to that of the wild-type glucocorticoid receptor, which has previously been shown to interact with hsp90 in vitro. These results provide evidence for the action of hsp90 as a molecular chaperone by selectively recognizing destabilized proteins.

Non-cell-autonomous function of the Antirrhinum floral homeotic proteins DEFICIENS and GLOBOSA is exerted by their polar cell-to-cell trafficking.

In Antirrhinum majus, petal and stamen organ identity is controlled by two MADS-box transcription factors, DEFICIENS and GLOBOSA. Mutations in either of these genes result in the replacement of petals by sepaloid organs and stamens by carpelloid organs. Somatically stable def and glo periclinal chimeras, generated by transposon excision events, were used to study the non-cell-autonomous functions of these two MADS-box proteins. Two morphologically distinct types of chimeras were analysed using genetic, morphological and molecular techniques. Restoration of DEF expression in the L1 cell layer results in the reestablishment of DEF and GLO functions in L1-derived cells only; inner layer cells retain their mutant sepaloid features. Nevertheless, this activity is sufficient to allow the expansion of petal lobes, highlighting the role of DEF in the stimulation of cell proliferation and/or cell shape and elongation when expressed in the L1 layer. Establishment of DEF or GLO expression in L2 and L3 cell layers is accompanied by the recovery of petaloid identity of the epidermal cells but it is insufficient to allow petal lobe expansion. We show by in situ immunolocalisation that the non-cell-autonomy is due to direct trafficking of DEF and GLO proteins from the inner layer to the epidermal cells. At least for DEF, this movement appears to be polar since DEF acts cell-autonomously when expressed in the L1 cell layer. Furthermore, the petaloid revertant sectors observed on second whorl mutant organs and the mutant margins of petals of L2L3 chimeras suggest that DEF and GLO intradermal movement is limited. This restriction may reflect the difference in the regulation of primary plasmodesmata connecting cells from the same layer and secondary plasmodesmata connecting cells from different layers. We propose that control of intradermal trafficking of DEF and GLO could play a role in maintaining of the boundaries of their expression domains.

The myelin basic protein‐specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential Vβ8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment

In the Lewis rat, myelin basic protein (MBP)-specific, encephalitogenic T cells preferentially recognize sequence 68-88, and use the V beta 8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP-specific T cell repertoire, we reconstituted severe-combined immunodeficient (SCID) mice with fetal (embryonic day 15-16) Lewis rat lymphoid tissue, and then isolated MBP-specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP-immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B1), and none by I-Ad of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68-88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP-specific T cell clones used a seemingly random repertoire of V beta genes without a bias for V beta 8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of V beta 8.2 was restored. The migration of liver-derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild-type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68-88 by MBP-specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.