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PML is a protein associated with discrete spherical structures within the nucleus of normal cells. A defect in PML expression is observed in acute promyelocytic leukaemia as a consequence of a chromosomal translocation involving the genes encoding PML and the alpha retinoic acid receptor (RAR alpha). Disruption of PML bodies also occurs during herpes simplex virus infection after the immediate early protein Vmw110 has become associated with PML bodies. In this study, we followed the fate of PML bodies in human fibroblasts during the course of a human cytomegalovirus (CMV) infection. Disruption of PML bodies was observed to be dependent on de novo CMV gene expression and to occur within 4 h post-infection, concomitant with the onset of CMV IE gene expression. Although a transient increase in the number of PML bodies could be observed in some cells, PML exists predominantly as a diffuse nuclear protein during both the early and late stages of CMV infection. Although the function of PML bodies is still uncertain, their disruption may be important for efficient herpes virus replication.

The promastigote surface antigen 2 (PSA-2) complex comprises a family of antigenically similar polypeptides of M(r) 96,000, 80,000 and 50,000, anchored to the membrane with glycosylphosphatidylinositol. Although PSA-2 was initially detected only in promastigotes, Northern blot analysis indicated that mRNA transcripts are also present in amastigotes. Unlike the situation in promastigotes, where at least four major transcripts (2.6-5.3 kb) were detected, only one major (2.6 kb) and two minor transcripts were present in amastigotes. A cDNA clone encoding a member of the PSA-2 family expressed in amastigotes was isolated using DNA probes. The predicted protein sequence of M(r) 40,000 is distinct from promastigote sequences, but shows significant similarity to previously described members of the family from L major and L amazonensis. Antibodies to the carboxyl terminal sequence conserved in all L major PSA-2 studied to date, as well as antibodies affinity purified on the amastigote cDNA-derived polypeptide recognized a major M(r) 50,000 amastigote polypeptide. Immuno-electron microscopy localized both promastigote and amastigote PSA-2 to the cell surface. The expression of PSA-2 polypeptides during the transformation of amastigotes into promastigotes was ordered in a time-dependent manner, with the promastigote M(r) 80000 polypeptide appearing first, followed by the M(r) 96000 polypeptide. In contrast to the glycosylphosphatidylinositol anchor of promastigote PSA-2, which could be hydrolysed by phosphatidylinositol-specific phospholipase C, the amastigote form was resistant to this enzyme.

Autoimmune gastritis, induced by day-3 thymectomy of BALB/c mice, is a destructive CD4+ T cell-mediated disease characterized by leukocyte infiltrates in the gastric mucosa, loss of parietal and chief cells and anti-gastric H/K ATPase autoantibodies. Our previous studies have indicated that a T cell response to the H/K ATPase beta subunit is required for the onset of autoimmune gastritis (Alderuccio, F., Toh, B. H., Tan, S. S., Gleeson, P. A. and van Driel, I. R., J. Exp. Med. 1993. 178: 419). To determine whether a response to the beta subunit autoantigen is alone sufficient to induce autoimmunity, or whether other tissue-specific factors are required, we have generated transgenic mice expressing the gastric H/K ATPase beta subunit in beta islet cells of the pancreas (RIP-H/K beta). RIP-H/K beta mice developed autoimmune gastritis and insulitis after day-3 thymectomy. Significantly, insulitis, observed as a peri-islet infiltrate, was only detected in thymectomized mice with autoimmune gastritis. There was no apparent immune destruction of the pancreas as insulitis did not progress to invasion of the islets or diabetes. Double transgenic mice, expressing the gastric H/K ATPase beta subunit in the thymus and in the pancreas, were protected from both gastritis and insulitis after day-3 thymectomy. Therefore, insulitis in the RIP-H/K beta mice appears to be dependent on a T cell response to the H/K ATPase beta subunit. This is the first example where an organ-specific initiating autoantigen has been expressed in another peripheral tissue. Autoimmune destruction in the stomach, but not the pancreas, indicates that tissue-specific factors play a fundamental role in the development of organ-specific autoimmunity.

This overview describes the spatial distribution of several enzymatic machineries and functions in the interphase nucleus. Three general observations can be made. First, many components of the different nuclear machineries are distributed in the nucleus in a characteristic way for each component. They are often found concentrated in specific domains. Second, nuclear machineries for the synthesis and processing of RNA and DNA are associated with an insoluble nuclear structure, called nuclear matrix. Evidently, handling of DNA and RNA is done by immobilized enzyme systems. Finally, the nucleus seems to be divided in two major compartments. One is occupied by compact chromosomes, the other compartment is the space between the chromosomes. In the latter, transcription takes place at the surface of chromosomal domains and it houses the splicing machinery. The relevance of nuclear organization for efficient gene expression is discussed.

Stomach acid is produced by parietal cells and secreted into the gastric lumen for digestive and protective purposes. The gastric parietal cell has a highly developed acid secretory machinery that can maintain the pH of the gastric lumen at around pH 1 (Forte and Soil, 1993; Sachs et al., 1992; Faller et al., 1993; Rabon and Reuben, 1990). The central role of the gastric H/K ATPase in the secretion of gastric HC1 is undisputed. The protein is located in the membranes of the parietal cell tubulovesicles and secretory canaliculi. The mechanism of action and structure of the gastric H/K ATPase have been extensively reviewed in this volume and elsewhere. The protein is composed of two subunits, a catalytic α and a β subunit. The existence of the a subunit has been known for some time. The protein is 95 kDa and has a large number of membrane spanning domains (probably 8) (Sachs et al., 1992; Green and Stokes, 1992). The ps subunit was only recently discovered in a number of laboratories (Okamoto et al., 1990, Reuben et al. 1990, Shull, 1992, Toh et al, 1990). The 35 kDa protein core is highly glycosylated with N-linked glycans which may terminate in poly-N-acetyllactosamine structures (Callghan et al. 1990, 1992). The β subunit appears to be essential for membrane insertion, may contain intracellular trafficking signals and is required for functional activity (Horisberg et al., 1991; Gottardi and Caplan, 1993; Sachs et al., 1992)

Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5′-triphosphate and digoxigenin-11-deoxyuridine 5′-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.

The immune system is programmed to defend the body against invading microbes by recognising antigens displayed on the surface of the organisms. At the same time, the immune system should not respond to “self” antigens. This ability to discriminate between “foreign” and “self” is known as immunological tolerance. Autoimmune disease develops when “tolerance” to self antigens breaks down. Currently, little is known about how tolerance can be broken, although it is generally thought that this may occur in genetically susceptible individuals exposed to certain environmental “triggers”. Furthermore, little is known about the early events which initiate the immune response to self antigens and in particular the way in which self antigens are recognised by T cells to activate the self-destructive autoimmune process. While in full blown autoimmune disease, the autoimmune response is clearly recognising a number of different antigenic determinants, it is possible that the early response may be directed towards one or only a few antigenic determinants.

Autoimmune gastritis is an excellent example of the organ-specific autoimmune diseases. The gastric lesion, characterized by loss of parietal and chief cells from the mucosa and submucosal lymphocytic infiltration, is the underlying basis for pernicious anaemia, the most common cause of vitamin B12 deficiency in whites of northern European origin (Gleeson and Toh, 1991; Toh et al, 1992). The gastric parietal cell appears to be the principal cell targeted since autoantibodies to parietal cells and to intrinsic factor (a parietal cell secretory product) are found in most patients with pernicious anaemia and parietal cell autoantibody titres correlate with severity of gastritis. These parietal cell autoantibodies are directed against the catalytic α and the glycoprotein β-subunit of the gastric H+/K+-ATPase (proton pump), the enzyme responsible for gastric acid secretion (Karlsson et al, 1988; Goldkorn et al, 1989; Toh et al, 1990). To investigate the immunopathogeriesis of autoimmune gastritis, we have adopted an experimental model of autoimmune gastritis induced by neonatal thymectomy carried out 2–4 days after birth in BALB/c mice. The murine disease is an ideal model since it displays the major features of the human disease with loss of parietal and chief cells accompanied by submucosal immunocytic infiltration and circulating parietal cell autoantibodies (Fukuma et al, 1989). These parietal cell autoantibodies also target the α and β-subunits of the proton pump (Jones et al, 1991). The murine disease appears to be cell-mediated, since the disease can be transferred by CD4+ T cells but not by autoantibodies (Sakaguchi et al, 1985).

Autoimmune gastritis results from the breakdown of T cell tolerance to the gastric H(+)/K(+) ATPase. The gastric H(+)/K(+) ATPase is responsible for the acidification of gastric juice and consists of an α subunit (H/Kα) and a β subunit (H/Kβ). Here we show that CD4(+) T cells from H/Kα-deficient mice (H/Kα(-/-)) are highly pathogenic and autoimmune gastritis can be induced in sublethally irradiated wildtype mice by adoptive transfer of unfractionated CD4(+) T cells from H/Kα(-/-) mice. All recipient mice consistently developed the most severe form of autoimmune gastritis 8 weeks after the transfer, featuring hypertrophy of the gastric mucosa, complete depletion of the parietal and zymogenic cells, and presence of autoantibodies to H(+)/K(+) ATPase in the serum. Furthermore, we demonstrated that the disease significantly affected stomach weight and stomach pH of recipient mice. Depletion of parietal cells in this disease model required the presence of both H/Kα and H/Kβ since transfer of H/Kα(-/-) CD4(+) T cells did not result in depletion of parietal cells in H/Kα(-/-) or H/Kβ(-/-) recipient mice. The consistency of disease severity, the use of polyclonal T cells and a specific T cell response to the gastric autoantigen make this an ideal disease model for the study of many aspects of organ-specific autoimmunity including prevention and treatment of the disease.

Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.

The glucocorticoid receptor (GR) is often described to be localized in the cytoplasm in the absence of hormone and to translocate to the nucleus upon binding of the hormone. This apparently different behavior of the GR compared to that of other members of the steroid receptor superfamily is unexpected because similarities in the molecular structures of steroid hormone receptors would predict similarities in their working mechanisms. The absence of the unliganded GR from the nuclear compartment may be due to an artefactual redistribution, occurring during the immunocytochemical procedure. We systematically studied the effects of various fixation and permeabilization procedures on the distribution of the GR in hepatoma cells that were incubated in steroid-free or steroid-containing medium. Immunofluorescent labeling of the GR in formaldehyde-fixed and detergent-permeabilized cells resulted in the almost complete absence of immunoreactivity of cells when the receptor was in its unliganded form, whereas the liganded receptor was detected mainly in the nucleus. On the other hand, labeling of cryosectioned glutaraldehyde/formaldehyde-fixed cells demonstrated that receptor antigenicity is present in the nucleus in the absence as well as the presence of steroid. We conclude that the results obtained with the cryosectioning procedure reflect the receptor distribution in the living cell, since glutaraldehyde fixation of the cells prevented the washout of loosely bound receptor. The predominantly nuclear location of the unliganded GR in hepatoma cells and the absence of changes in distribution after steroid stimulation imply that the nuclear translocation model is not true for the GR.

The alpha and beta subunits of the gastric H+/K(+)-ATPase (proton pump) have been identified as the major molecular targets of parietal cell autoantibodies associated with pernicious anaemia and with murine experimental autoimmune gastritis (EAG) induced by neonatal thymectomy. Recent studies with EAG suggest that the mechanisms of peripheral tolerance and autoimmunity to extrathymic autoantigens are mediated by subsets of "regulator" and "effector" CD4+ T cells, respectively. The persistence of "effector" CD4+ autoreactive T cells in the periphery may be a direct consequence of the delayed developmental expression of the target autoantigen. We hypothesize that cytokines produced by the "regulator" T cells prevent the clonal expansion of the "effector" autoreactive T cells, and that neonatal thymectomy induces organ-specific autoimmunity in genetically susceptible individuals by the reduction of the "regulator" T cell population.

Background & Aims Gastric cancer is the second most common cause of cancer-related mortality worldwide, mainly as a result of late-stage detection. Interleukin (IL)-11 is a multifunctional cytokine reported to be up-regulated in human gastric cancer. Methods We investigated the importance of IL-11 in gastric cancer progression by examining its role in a variety of mouse gastric tumor models, as well as in nonneoplastic and tumor tissues taken from gastric cancer patients. We then determined the transcriptional and translational outcomes of IL-11 overexpression in normal gastric mucosa and identified a novel gene signature important early in the progression toward gastric tumorigenesis. Results IL-11 was up-regulated significantly in 4 diverse mouse models of gastric pathology as well as in human biopsy specimens adjacent to and within gastric cancer. Removal of IL-11 co-receptor α significantly reduced HKβ-/- mouse fundic hyperplasia and ablated gp130 757F/F mouse tumorigenesis. Exogenous IL-11 but not IL-6 activated oncogenic signal transducer and activator of transcription-3, and altered expression of novel proliferative and cytoprotective genes RegIII-β, RegIII-γ, gremlin-1, clusterin, and growth arrest specific-1 in wild-type gastric mucosa, a gene signature common in gp130 757F/F and HKβ-/- tumors as well as nonneoplastic mucosa of gastric cancer patients. One week of chronic IL-11 administration in wild-type mice sustained the gene signature, causing pretumorigenic changes in both antrum and fundus. Conclusions Increased gastric IL-11 alters expression of proliferative and cytoprotective genes and promotes pretumorigenic cellular changes.

We describe a method for immunogold staining of nuclear matrix proteins using ultra-small gold particles. The nuclear matrix of HeLa cells is obtained by two fractionation steps: (a) cell permeabilization with Triton X-100 to isolate the cytoskeleton, and (b) nuclease digestion followed by an incubation in 0.25 M ammonium sulfate to isolate the nuclear matrix. To prevent redistribution of internal matrix proteins during nuclear matrix preparation, pre-fixation with 0.1% acrolein was performed. Under this condition up to 80% of protein and 90% of DNA and RNA could be removed on nuclear matrix isolation, without redistribution of internal nuclear matrix proteins. For immunogold labeling, 1-nm gold probes appeared to be required to obtain optimal penetration into the nucleus. These particles can be visualized after silver enhancement. After gold labeling the matrices are stained, embedded in Epon, and ultra-thin sections are prepared for examination in the electron microscope. The applicability of this method is examplified by the localization of a 125 KD internal nuclear matrix protein and the lamins A and C in nuclear matrix preparations of HeLa cells.

In situ nick-translation allows the visualization of nuclease-sensitive chromatin regions in interphase nuclei. We have analyzed the three-dimensional (3-D) distribution of DNase I-sensitive regions of chromatin in nuclei from mouse P19 embryonal carcinoma cells by making optical sections using confocal scanning laser microscopy. In undifferentiated as well as embryonal carcinoma cells differentiated in vitro, DNase I-sensitive regions of chromatin are observed as discrete spots in the nucleus. These spots represent clusters of DNase I-sensitive sites. By optical sectioning, we show that these spots are preferentially, but not exclusively, localized at the nuclear periphery. No differences were observed in the spatial distribution of DNase I-sensitive sites in P19 EC cells or the differentiated P19 END-2 cells. Furthermore, we did not observe differences in the distribution of DNase I-sensitive chromatin regions during the cell cycle. These findings indicate, at least for P19 mouse embryonal carcinoma cells and their differentiated derivative END-2, that the compartmentalization of DNase I-sensitive chromatin regions is a general characteristic of the nucleus, independent of cell cycle stage or differentiation state. Since evidence has been presented that DNase I-sensitive sites are associated with actively transcribed chromatin, our results indicate that active transcribing chromatin is compartmentalized, preferentially in the periphery of the nucleus.

In developing Dictyostelium discoideum cells, binding of cyclic AMP to the chemotactic receptor has been shown to oscillate. These oscillations represent cycles of activation, adaptation and deadaptation of the cyclic AMP receptor system. We studied the molecular basis of these oscillatory changes in cyclic AMP receptor binding. We developed a rapid method of lysing cells during the course of the oscillations. This method guaranteed good preservation of ligand binding properties of the cyclic AMP receptor. We found that oscillations in cyclic AMP binding resulted from changes in receptor affinity. The total number of receptors did not significantly change during oscillations. Our experiments also showed that both GTP and GDP abolished oscillations in receptor binding completely, presumably by acting via a G protein. The guanine nucleotides reduced the affinity of the receptor at all time-points of the oscillation cycle to the minimal, i.e. adapted, level. We conclude that the cyclic process of activation, adaptation and de-adaptation in D. discoideum, at cyclic AMP receptor level, involves changes in receptor-G protein interaction. During adaptation, the affinity of the cyclic AMP receptor decreases and the receptor becomes insensitive to guanine nucleotides.

The peroxisomal diseases can be divided into three categories: 1) diseases in which morphologically distinguishable peroxisomes are virtually absent (Zellweger syndrome; infantile Refsum disease; Hyperpipecolic Acidaemia; neonatal Adrenoleukodystrophy); 2) diseases in which peroxisomes are present but several peroxisomal functions are impaired (rhizomelic Chondrodysplasia punctata; Zellweger-like syndrome?); and 3) diseases in which a single peroxisomal function is impaired. We have used complementation analysis after somatic cell fusion in order to investigate the genetic relationship between diseases in category 1. The activity of acyl-CoA: dihydroxyacetonephosphate acyltransferase, which is deficient in these diseases and in rhizomelic Chondrodysplasia punctata, was used as an index of complementation. The cell lines studied, all of which complemented with rhizomelic Chondrodysplasia punctata, could be divided into at least 4 and possibly 5 complementation groups. This indicates that at least 5 and possibly 6 genes are involved in the assembly of peroxisomes. One of the complementation groups is comprised of cell lines from patients with the Zellweger syndrome, infantile Refsum disease and Hyperpipecolic Acidaemia. Thus mutations in the same gene can lead to clinically distinguishable diseases. On the other hand, the Zellweger cell lines studied fall into 3 complementation groups and the infantile Refsum disease cell lines into 2 groups. Thus mutations in different genes can lead to the same clinical phenotype. Fusion of complementary cell lines lacking morphologically distinguishable peroxisomes leads to assembly of peroxisomes, which can be monitored by measuring particle-bound catalase biochemically or by immunofluorescence. In two combinations of cell lines assembly of peroxisomes was rapid and cycloheximide insensitive. Thus the components required for peroxisome assembly must be present in a stable form in the parental cell lines, at least one of which must contain peroxisomal ghost-like structures.

Microair bubbles in a bloodstream are diverted from the bloodstream in a diversion chamber by ultrasonic energy, and are collected in a blood-filled stasis column where they can be accurately measured by a bubble detector and then vented to atmosphere. Bubbles of different sizes can be separated into different stasis columns for enhanced measurement.

Cells from Dictyostelium discoideum carry chemotactic cAMP receptors on their surface. Kinetic studies have revealed the existence of two slowly dissociating, high affinity receptor forms (SS and S) and one or more fast dissociating, low affinity forms (F) (Van Haastert, P.J.M., and De Wit, R.J.W. (1984) J. Biol. Chem. 259, 13321-13328). We have studied the interaction of these different cAMP-receptor types with a detergent-insoluble membrane residue. Isolated D. discoideum membranes were extracted with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), which was previously shown to be the only detergent in the presence of which cAMP receptor binding is completely preserved (Janssens, P. M. W., and Van Driel, R. (1986) Biochim. Biophys. Acta 885, 91-101). The protein composition of the CHAPS-insoluble membrane residue appeared to be similar to that of the Triton X-100-insoluble membrane skeleton. Cyclic AMP binding studies revealed a specific association of the slowly dissociating cAMP receptors (SS and S forms) with this CHAPS-insoluble residue. All fast dissociating (F type) receptors were solubilized by CHAPS. GTP induced a transition of 75% of the SS and S receptors to faster dissociating forms. This transition was accompanied by the release of an equal number of receptors from the residue. These effects of GTP required that the cAMP receptor was occupied, and were completely reversible. After removal of the guanine nucleotide SS and S type receptors reappeared, bound to the residue, with a t1/2 of 5-10 min at 0 degrees C. We conclude that a detergent-insoluble membrane residue is involved in signal transduction via the chemotactic cAMP receptor. Both receptor occupation and a guanine nucleotide binding protein control receptor-residue interaction.

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma (EC) cells was compared with that of clonal derivatives of P19 EC differentiated in vitro, and with that of P19 EC cells induced to differentiate with retinoic acid (RA). Several major differences in nuclear matrix protein composition were found between the cell lines tested. Some polypeptides were found to occur only in EC cells, whereas others proved to be restricted to one or more of the differentiated derivatives. During RA treatment of EC cells a transient expression of some matrix proteins was observed. Several new proteins appeared, and others disappeared. Our data indicate that the protein composition of the nuclear matrix is a sensitive gauge for the differentiation state of cells.

When plus and minus mating type gametes of Chlamydomonas eugametos were mixed, a rapid transient increase in the amount of cAMP was observed with a maximum at 20 s after the start of the sexual agglutination reaction. The transient increase only occurred within the cells and was also exhibited when cell suspensions of single mating type were presented with isolated flagella of the other mating type. Cyclic AMP-dependent protein kinase and cyclic AMP-phosphodiesterase activities were found in cell homogenates. Since the rise in cAMP concentration preceded all known morphological and physiological changes in the cells that prepare them for fusion, it might be a primary response, induced by sexual agglutination.

We describe herein the use of reversed-phase high-performance liquid chromatography coupled with the novel application of short (10 cm or less) microbore columns (2 mm internal diameter) to fractionate and purify a number of tryptic peptides generated from approximately 200 pmol purified murine transferrin receptor. The use of reversed-phase microbore columns permits the recovery of submicrogram amounts of purified polypeptides in high yield (greater than 90%) in small eluent volumes (20-60 microliter). In this manner, purified polypeptides can be loaded directly onto the gas-phase sequencer without further manipulation. This procedure avoids sample loss, which frequently occurs with other forms of concentration (e.g. lyophilization, evaporation). The application of second-order-derivative ultraviolet spectroscopy, using a diode array detector, for the analysis of aromatic aminoacid-containing peptides in complex tryptic digests is described. N-terminal amino acid sequence analyses were performed on six tryptic peptides, yielding 105 unique assignments; this corresponds to approximately 14% of the molecule. A comparison of this amino acid sequence information with the primary structure of human transferrin receptor deduced from the mRNA sequence [Nature (Lond.) 311, 675-678 (1984); Cell 39, 267-274 (1984)] reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.

Protein phosphatase activities in developing Dictyostelium discoideum cells were investigated. Substrates were prepared by phosphorylation of histone H2b and kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) using cAMP-dependent protein kinase. Two histone phosphatase activities (Mr 170 000 and 520 000) and one kemptide phosphatase activity (Mr 230 000) were found in the cytosolic cell fraction. Histone phosphatase was also present in the particulate fraction, kemptide phosphatase was not. All phosphatase activities were present throughout development. No differences in protein phosphatase activities were found in prespore and prestalk cells. A heat-stable factor which inhibits the particulate and both soluble histone phosphatase activities was isolated.

Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.

Previous studies of Europe-Finner & Newell indicated that in amoebae of Dictyostelium discoideum, signal transduction used for chemotaxis to cyclic AMP involved transient formation of inositol tris- and polyphosphates. Evidence was also presented for the involvement of a GTP-binding G-protein. Here we report evidence for the involvement of a ras gene product in the D. discoideum inositol phosphate pathway. Use was made of strains of Dictyostelium transformed with a wild-type D. discoideum ras gene (ras-Gly12) or a mutant form of the gene (ras-Thr12). Experiments using separation of soluble inositol phosphates by Dowex anion-exchange resin chromatography indicated that cells transformed with the wild-type ras-Gly12 gene were unaffected in their basal levels of inositol polyphosphates and in the inositol phosphates formed in response to stimulation with the chemotactic agent cyclic AMP. In contrast, cells transformed with the mutant ras-Thr12 gene showed a basal level of inositol polyphosphate that was several-fold elevated over the controls and stimulation of these cells with cyclic AMP produced only a small further elevation. When the inositol phosphates were analysed by h.p.l.c. it was found that the basal level of inositol 1,4,5-trisphosphate was raised three- to fivefold in the ras-Thr12 strain compared to the strain transformed with ras-Gly12, and that inositol hexakisphosphate (which was found to be present in large amounts relative to other inositol phosphates in D. discoideum cells) was also raised to a similar extent in the ras-Thr12-transformed cells. We propose that the Dictyostelium ras gene product codes for a regulatory protein involved in the inositol phosphate chemotactic signal-transduction pathway.

We investigated whether cyclic AMP is an essential extracellular stimulus for the differentiation of prespore cells in slugs of D. discoideum. A local reduction of the extracellular cAMP level inside the slug was induced by implantation of cAMP-phosphodiesterase (cAMP-PDE)-coated spheres in intact slugs. This treatment caused the disappearance of prespore antigen in the vicinity of the sphere. A general reduction of extracellular cAMP levels in slugs, induced by submerging slugs in 0·25i.u.ml−1 cAMP-PDE, reduced the proportion of prespore cells from 66 % to 15 %, without affecting slug morphology. The cAMP-PDE-induced dedifferentiation of prespore cells was counteracted by cAMP and was not due to the production of the hydrolysis product 5′AMP, but to the reduction of extracellular cAMP levels. We conclude that extra-cellular cAMP is the major morphogenetic signal for the differentiation of prespore cells in the multicellular stages of D. discoideum development and we present a working hypothesis for the generation of the prestalk/ prespore pattern during multicellular development.

The PC-1 protein is a membrane glycoprotein that is selectively expressed on the surface of antibody-secreting cells. Previous work has shown that it consists of two apparently identical disulfide-bonded polypeptides, each of molecular weight approximately 120,000. We now describe the sequence of PC-1 mRNA and protein. The PC-1 protein is shown to consist of 905 amino acids and to have an uncommon transmembrane orientation. The NH2-terminal 58 residues are intracellular and the COOH-terminal 826 residues are extracellular. A cysteine-rich region of 85 amino acids lies adjacent to the extracellular surface of the membrane and appears to have arisen by exon duplication. In common with other membrane glycoproteins with this orientation, there is no obvious signal sequence other than the transmembrane segment. The PC-1 protein therefore has an overall structure and membrane orientation that resembles those of the transferrin receptor, the asialoglycoprotein receptor, and the Ia invariant chain.

Aggregating Dictyostelium discoideum cells possess highly specific receptors for the chemoattractant cAMP on their cell surface. Isolated membranes as well as intact cells are shown to contain a large number of latent cAMP receptors. These are reversibly unmasked in the presence of a high salt concentration (0.1–2 M) or in the presence of millimolar concentrations of Ca2+.

Dictyostelium discoideum cells contain a single ras gene (Dd-ras) that is highly homologous to mammalian ras genes. Cell transformation with a vector carrying a ras gene with a (glycine----threonine) missense mutation at position 12 causes an altered morphogenesis. Extracellular cAMP signals regulate morphogenesis and induce chemotaxis and the activation and subsequent desensitization of adenylate and guanylate cyclase. cAMP signal transduction was investigated in Dd-ras-transformed cells. Transformants that overexpress the mutated Dd-ras-Thr12 gene show normal activation and desensitization of adenylate cyclase and normal activation of guanylate cyclase. However, cAMP induces a stronger desensitization of guanylate cyclase stimulation in the Dd-ras-Thr12 transformant than in transformants overexpressing the Dd-ras-Gly12 wild-type gene or in untransformed cells. This effect was correlated with a reduced chemotactic sensitivity of the transformant expressing the mutated Dd-ras-Thr12 gene.

The ligand binding domain of the low density lipoprotein receptor consists of seven cysteine-rich repeats of approximately 40 amino acids each. These repeats, which are located at the NH2 terminus of the protein, are homologous to sequences in complement components C8 and C9. To determine the role of the first repeat (amino acids 2-42), we prepared two plasmids containing expressible low density lipoprotein receptor cDNAs. The first plasmid, p delta R1, lacks only the nucleotides encoding the first repeat. It produced a receptor that bound and internalized lipoproteins and recycled to the cell surface with the same efficiency as the normal receptor. This deleted receptor failed to bind two monoclonal antibodies, IgG-C7 and IgG-15C8, which were shown previously to react with the ligand-binding domain. The second plasmid, pR1, encodes a markedly truncated protein whose extracellular domain consists of the first repeat joined to the transmembrane and cytoplasmic domains. This protein bound the two monoclonal antibodies with the same affinity as the normal receptor, but failed to bind lipoproteins. Binding of IgG-15C8 to the normal receptor and the pR1-encoded protein was Ca2+-dependent, indicating that the first repeat binds Ca2+. We conclude that repeats 2-6 in the ligand-binding domain are sufficient for binding lipoproteins and that the first repeat is highly immunogenic, but is not required for lipoprotein binding.

The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.

Dictyostelium discoideum cells contain kinetically distinguishable surface cAMP receptors. Both GTP and GDP lower the receptor affinity by inducing conversion of slowly dissociating sites to fast dissociating sites, presumably by binding to a N-protein [(1985) Mol. Cell. Biochem. 67, 119-124, and (1986) Biochemistry 25, 1314-1320]. In this paper we show that treatment of isolated membranes with cAMP-dependent protein kinase abolished the GTP-induced receptor transition, but not the one induced by GDP. The effect of GTP on the receptor kinetics could be restored by treatment of the membranes with alkaline phosphatase. These results indicate that in D. discoideum membranes phosphorylation of a signal-transduction component reversibly abolishes the interaction of the cAMP receptor with the N GTP complex, but not that with the N SDP complex.

The 50-residue cytoplasmic domain of the low density lipoprotein receptor (amino acids 790-839) directs the receptor to coated pits, thereby facilitating rapid endocytosis of bound low density lipoprotein. To determine the structural features required for this targeting, we produced 24 mutations in the cytoplasmic domain through use of oligonucleotide-directed mutagenesis. The first 22 amino acids of the cytoplasmic domain (residues 790-811) are sufficient for rapid internalization. The amino acid at position 807 is especially critical. Aromatic residues (tyrosine, phenylalanine, or tryptophan) at this position allow rapid internalization. Charged or uncharged aliphatic residues do not substitute. Although the requirements at the neighboring positions (806 and 808) are less stringent, the insertion of proline at position 806 is detrimental. These specificities suggest that the juxtamembranous region of the cytoplasmic domain participates in protein:protein interactions that allow the low density lipoprotein receptor to cluster in coated pits.

When the low density lipoprotein (LDL) receptor was solubilized from bovine adrenal cortex membranes and subjected to electrophoresis in the absence of reducing agents, a disulfide-bonded dimeric species was demonstrated. Formation of these covalent bonds was blocked when the tissue was homogenized in the presence of sulfhydryl alkylating agents, indicating that the native receptor was self-associated noncovalently and that the disulfide bond formation occurred only after homogenization. The disulfide-linked dimers were disrupted and the receptor was restored to a monomeric form when inside-out adrenal vesicles were treated with trypsin, suggesting that the disulfide bond formation involved the 50-amino acid cytoplasmic domain of the receptor. When the receptor was solubilized from bovine adrenal cortex membranes and then purified by ion exchange and affinity chromatography, it could be covalently coupled into dimers and trimers in the presence of bivalent cross-linking agents. Receptor dimers could also be demonstrated by chemical cross-linking of intact cells that were transfected with an expressible cDNA encoding the normal human LDL receptor. Dimer formation was markedly reduced in transfected cells expressing mutated cDNAs that had premature termination codons at positions 792, 807, and 812, which produced shortened receptors that retained 2, 17, and 22 of the original 50 amino acids of the cytoplasmic domain, respectively. The first two mutant receptors, which did not form oligomers, did not enter coated pits and were not rapidly internalized by cells. However, the mutant receptor that terminates at position 812 was internalized normally even though oligomer formation was greatly reduced. Moreover, a mutant receptor with a cysteine substituted for a tyrosine at position 807, which internalized very slowly, showed a normal susceptibility to chemical cross-linking. Deletion of external domains of the LDL receptor, including the epidermal growth factor homology region and the O-linked sugar domain, did not alter susceptibility to chemical cross-linking. We conclude that the cytoplasmic domain of the LDL receptor is responsible both for self-association into oligomers and for clustering in coated pits, but the available data do not establish a causal connection between these two events.

Folate deaminase released from cells of Dictyostelium discoideum is heterogeneous with respect to molecular weight and stability at 60 degrees C. The most heat-stable component isoelectrofocuses in a broad band at approx. pH 6. The Km value of this component for folate is approx. 7 x 10(-7)M and Mr approx. 40 000. The major portion if not all of the deaminase binds to immobilized concanavalin A and lentil lectin. Extracellular folate deaminase has a pH-optimum of approx. pH 6.0. This is higher than that of lysosomal enzymes, which are also glycoproteins released into the extracellular medium.

1. 1. Reintroduction of monovalent copper into apo-α-hemocyanin of Helix pomatia leads to the complete return of the properties of native α-hemocyanin. 2. 2. The copper atoms in hemocyanin are required for the reassociation of tenth and twentieth molecules. 3. 3. In the reintroduction process, the copper atoms are randomly distributed among the empty binding sites of apo-α-hemocyanin. All copper-binding sites are equivalent and grouped in pairs; each pair forms a functional group. 4. 4. Upon storage for a few months, part of the copper atoms in every hemocyanin molecule are oxidized to Cu 2+ . This affects the oxygenation properties. 5. 5. The oxygenation curves of fresh α- and β-hemocyanin are nonhyperbolic at alkaline pH values in the presence of Ca 2+ and Mg 2+ . The influence of the pH on the oxygenation curves of α- and β-hemocyanin shows marked differences.

The ligand binding domain of the low density lipoprotein receptor consists of seven cysteine-rich repeats of approximately 40 amino acids each. These repeats, which are located at the NH2 terminus of the protein, are homologous to sequences in complement components C8 and C9. To determine the role of the first repeat (amino acids 2-42), we prepared two plasmids containing expressible low density lipoprotein receptor cDNAs. The first plasmid, p delta R1, lacks only the nucleotides encoding the first repeat. It produced a receptor that bound and internalized lipoproteins and recycled to the cell surface with the same efficiency as the normal receptor. This deleted receptor failed to bind two monoclonal antibodies, IgG-C7 and IgG-15C8, which were shown previously to react with the ligand-binding domain. The second plasmid, pR1, encodes a markedly truncated protein whose extracellular domain consists of the first repeat joined to the transmembrane and cytoplasmic domains. This protein bound the two monoclonal antibodies with the same affinity as the normal receptor, but failed to bind lipoproteins. Binding of IgG-15C8 to the normal receptor and the pR1-encoded protein was Ca2+-dependent, indicating that the first repeat binds Ca2+. We conclude that repeats 2-6 in the ligand-binding domain are sufficient for binding lipoproteins and that the first repeat is highly immunogenic, but is not required for lipoprotein binding.

The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.

Folate stimulation is known to enhance Dictyostelium discoideum differentiation. During early differentiation, D. discoideum cells possess two classes of folate receptors which can be distinguished by their difference in specificity (R. J. W. de Wit, FEBS Lett. 150, 445-448, 1982). We investigated the type of receptor by which folate affects cell differentiation. Two independently regulated developmental markers were used: the extracellular phosphodiesterase-inhibitor system and cell-surface phosphodiesterase activity. Our results indicate that the major effect of folate on development is mediated by the folate-specific receptor. The nonspecific folate receptor was only involved in a minor, transient enhancement of the extracellular phosphodiesterase activity very early in development.

The presence and intracellular localization of peroxisomal integral membrane proteins (PMP) were investigated in liver and cultured skin fibroblasts from control subjects and patients with the Zellweger syndrome and related disorders in which peroxisomes are virtually absent. Immunoblotting experiments showed that 22, 36 and 69 kDa PMPs were present and were confined to the membranous fraction both in the control liver and in the livers from the Zellweger patients. The 22 and 36 kDa PMPs were present in significantly lower amounts in the patients' livers than in the control liver. A reduced amount of the 69 kDa PMP was found in liver from one Zellweger but not in liver from another. The subcellular localization in fibroblasts of catalase and the 69 kDa PMP was studied by indirect immunofluorescence. A characteristic punctate fluorescence was seen in control cells incubated with either anti-(catalase) or with anti-(69 kDa PMP). Incubation of mutant cells with anti-(catalase) resulted in a diffuse fluorescence, whereas with anti-(69 kDa PMP) fluorescent particles were visualized which, in some cell lines, were larger and fewer in number than in control cells. Cryosections of control and mutant cells were examined by electron microscopy using immunogold labeling. Control cells contained small structures consisting of a single membrane enclosing a homogeneous matrix; the membranes reacted with anti-(69 kDa PMP) and the matrix with anti-(catalase). The mutant cell lines contained spherical or ellipsoidal structures whose membranes reacted with anti-(69 kDa PMP); no labeling was observed with anti-(catalase). We conclude that peroxisomal ghosts, the membranes of which contain the 69 kDa PMP, are present in peroxisome-deficient cell lines from all complementation groups studied so far.

The two exocyclic oxygen atoms at phosphorus of cAMP have been replaced by a sulfur atom or by a dimethylamino group. These substitutions introduce chirality at the phosphorus atom; therefore, two diastereoisomers are known for each derivative: (SP)-cAMPS, (RP)-cAMPS, (SP)-cAMPN(CH3)2, and RP-cAMPN(CH3)2. We have investigated the agonistic and antagonistic activities of these compounds in four cAMP-dependent reactions: activation of the cellular slime mold Dictyostelium discoideum via its cell surface cAMP receptor, and phosphorylation by cAMP-dependent protein kinases type I, type II (both mammalian enzymes), and type D (derived from D. discoideum). The results show that 1) the compounds (SP)-cAMPS and (SP)-cAMPN(CH3)2 are (mostly full) agonists for the four proteins. Half-maximal activation is at micromolar concentrations (0.8-7 microM). 2) (RP)-cAMPS is a full antagonist for the cell surface receptor and protein kinases type I and II, with apparent inhibition constants between 0.8 and 8 microM. This compound is a partial agonist for protein kinase type D, where it induces maximally 50% activation of the enzyme if compared with cAMP. 3) (RP)-cAMPN(CH3)2 is a full antagonist for the cell surface receptor, and for protein kinase type II. This compound is a partial agonist for protein kinase type I (at least 50% activation if compared with cAMP), and inactive for protein kinase type D. This derivative is at least 25-fold less active as an antagonist than (RP)-cAMPS. 4) The activity of mixtures of different concentrations of the antagonist (RP)-cAMPS with different concentrations of cAMP reveals that the compound is a competitive antagonist of cAMP at micromolar concentrations.