Biological and molecular characterization of cellular differentiation in Tetrahymena vorax: a potential biocontrol protozoan.

Tetrahymena vorax (T. vorax) is an indigenous fresh water protozoan with the natural biological potential to maintain a specific aquatic microbial flora by ingesting and eliminating specific microorganism. To investigate the molecular mechanisms controlling Tetrahymena vorax (T. vorax) cellular differentiation from a small-mouth vegetative cell to a voracious large-mouth carnivore capable of ingesting prey ciliates and bacteria from aquatic environments, we use DNA subtraction and gene discovery techniques to identify and isolate T. vorax differentiation-specific genes. The physiological necessity for one newly discovered gene, SUBII-TG, was determined in vivo using an antisense oligonucleotide directed against the 5' SUBII-TG DNA sequence. The barriers to delivering antisense oligonucleotides to the cytoplasm of T. vorax were circumvented by employing a new but simple procedure of processing the oligonucleotide with the differentiation stimulus, stomatin. In these studies, the antisense oligonucleotide down-regulated SUBII-TG mRNA expression, and blocked differentiation and ingestion of prey ciliates. The ability to down-regulate SUBII-TG expression with the antisense oligonucleotide suggests that the molecular mechanisms controlling the natural biological activities of T. vorax can be manipulated to further study its cellular differentiation and potential as a biocontrol microorganism.

Inhibition of beta-globin gene expression by 3'-azido-3'-deoxythymidine in human erythroid progenitor cells.

3'-Azido-3'-deoxythymidine (AZT) treatment in HIV-infected patients is limited by bone marrow suppression including neutropenia and anemia. Previous studies had shown a direct effect of high concentrations of this drug on globin gene expression in K-562 erythroleukemia cells. To better define the mechanism(s) of AZT-induced bone marrow toxicity, the present study evaluates these effects in more relevant human erythroid progenitor liquid cultures, because AZT is 100 times more toxic to human bone marrow cells than K-562 cells. At a clinically relevant concentration of 1 microM, AZT inhibited specifically erythroid cell growth by approximately 58% as compared with untreated cells. The percentage of cells synthesizing hemoglobin was decreased also by 47% in AZT-treated cells with beta-globin mRNA levels accounting for 0.27 pmol in treated cells as compared with 1.44 under control conditions while beta-actin levels remained unchanged. Under the same conditions, AZT inhibited the beta-globin chain synthesis by approximately 60% as compared with the control. Consistent with the data described above was the finding that a concentration as low as 0.1 microM of AZT decreased by almost 40% the binding level of the erythroid-specific transcription factor GATA-1. These findings demonstrate that AZT, at clinical relevant concentrations, specifically inhibits beta-globin gene expression in human erythroid progenitor liquid cell culture.

A novel stop codon mutation (X417L) of the ferrochelatase gene in bovine protoporphyria, a natural animal model of the human disease.

Protoporphyria (PP) is caused by a deficiency of ferrochelatase (FC) activity, which catalyzes the final step in the heme biosynthesis pathway. Bovine are the only species other than man with naturally occurring PP. For expression of the PP phenotype, two copies of the mutated gene are necessary in bovine, whereas one copy is sufficient in humans. We report the first potential disease-causing mutation in the bovine FC gene. The coding region of FC was sequenced from the liver tissue of protoporphyric and normal bovine. A transversion was identified at nucleotide position 1250 which changed the stop codon to leucine (TGA-->TTA) in the protoporphyric FC sequence. As a consequence, the mutant protein is predicted to have an additional 27 amino acids. To screen other bovine for the G-->T transversion, cDNAs from liver tissue of clinically and biochemically normal, and from heterozygous and homozygous affected animals were used for allele-specific polymerase chain reaction. Three normal animals had only the G allele, five affected animals had only the T allele, and three heterozygous animals had both the G and T alleles. These results support our hypothesis that this mutation causes PP in bovine.

Molecular cloning, sequence analysis, expression, and tissue distribution of suppressin, a novel suppressor of cell cycle entry.

Suppressin (SPN) is an inhibitor of cell proliferation that was originally identified and purified to homogeneity from bovine pituitaries (LeBoeuf, R. D., Burns, J. N., Bost, K. L., and Blalock, J. E. (1990) J. Biol. Chem. 265, 158-165). In this report we have cloned the full-length cDNA encoding rat SPN and have identified the tissue distribution of SPN expression. The cDNA of SPN is 1882 nucleotides with a 1488-base coding region and 55 and 339 nucleotides of 5'- and 3'-untranslated sequences, respectively. Northern gel analysis of rat pituitary mRNA showed a single hybridizing species at approximately 2 kilobases. Sequence analyses showed that the nucleotide and deduced amino acid sequences of SPN are novel and unrelated to any known vertebrate inhibitors of proliferation. However, the deduced amino acid sequence of SPN contains two domains that have extensive sequence identity with a recently cloned transcription activator in Drosophila, deformed epidermal autoregulatory factor-1 (DEAF-1, see Gross, C. T., and McGinnis, W. (1996) EMBO J. 15, 1961-1970) suggesting that SPN represents a vertebrate cognate of deformed epidermal autoregulatory factor-1. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses showed that the SPN mRNA and the SPN protein are expressed in every tissue examined including testis, spleen, skeletal muscle, liver, kidney, heart, and brain suggesting that SPN may be involved in the control of proliferation in a variety of cell types.

Negative regulatory molecules in the neuroendocrine and immune systems: suppressin as an example.

The early foundations of both neuroendocrinology and immunology were established by studies that linked the production, secretion, and action of circulating factors to the physiological state of an organism. These studies ultimately identified the cells of the neuroendocrine and immune systems as a rich source of such homeostatic regulatory molecules, and currently they are referred to as neuroendocrine hormones, peptides, and cytokines. More recently, two additional concepts have been added to this model. The first was that immune cells produce neuroendocrine hormones and peptides and that neuroendocrine cells produce cytokines. The second was the notion that both positive and negative factors control a variety of physiological processes. Recently, we have identified a new polypeptide negative regulator of cell proliferation that we have named suppressin (SPN). This negative regulatory molecule is also produced by both neuroendocrine and immune cells. The objective of this article is to provide an example of the biochemical, cellular, and molecular approaches used to characterize SPN and that could be used to characterize similar molecules from neuroendocrine and immune sources.

Suppressin: an endogenous negative regulator of immune cell activation.

We have recently identified a new suppressor molecule we named suppressin (SPN) that has all the characteristics of a global negative regulator of the immune system. SPN is a unique 63-kD monomeric polypeptide with a pI of 8.1 that is produced and secreted under basal conditions by murine splenocytes, human peripheral mononuclear cells, and hormone-secreting pituitary cells. The biological actions of SPN in vitro include the inhibition of mitogen-induced proliferation and immunoglobulin synthesis of lymphocytes and the suppression of interleukin-2-dependent CTLL-2 cell proliferation. In addition, SPN enhances natural killer cell activity by eliciting interferon-alpha and -beta synthesis and secretion. SPN effects are reversible, nontoxic, and require the continuous presence of exogenous SPN. T lymphocytes stimulated with concanavalin A or phytohemagglutinin are more sensitive to SPN (90% inhibition) than are lipopolysaccharide-stimulated B cells (60% inhibition). SPN arrests lymphocytes in the G0/G1 phase of the cell cycle after reduction of their RNA, protein and DNA synthesis, suggesting that SPN inhibits the processes required for G0 transition to G1. SPN is found intracellularly in all unstimulated lymphocyte subsets, monocytes, and in phytohemagglutinin-activated T lymphocytes immunopositive for the low affinity interleukin-2 receptor. These results suggest that SPN may be a major negative regulator of cell proliferation in the immune system. All SPN-producing cell types are also sensitive to SPN. Collectively, the results of these experiments provide the foundations for a model in which SPN regulates lymphocyte proliferation in an autocrine and/or paracrine manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of experimental autoimmune myasthenia gravis by manipulation of the immune network with a complementary peptide for the acetylcholine receptor.

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are caused, in part, by the production of autoantibodies against the main immunogenic region, amino acids 61-76, of the alpha chain of the acetylcholine receptor (AChR). Theoretically, induction of anti-idiotypic (Id) antibodies (Abs) should be a highly specific treatment for the disease by virtue of their potential ability to neutralize Abs to the AChR. We have tested this idea by attempting to evoke such anti-Id Abs by immunization with a peptide (termed RhCA 67-16) encoded by RNA complementary to the Torpedo AChR main immunogenic region and determining whether such treatment will prevent the development of EAMG. Immunization with RhCA 67-16, but not a control peptide termed PBM 9-1, was found to elicit the production of anti-Id Abs that blocked recognition of native Torpedo AChR by its Ab. This anti-Id Ab activity was ablated by incubation of the anti-RhCA 67-16 serum with RhCA 67-16, but PBM 9-1, prior to the assay for Ab binding to AChR. The anti-Id Ab-inducing activity of RhCA 67-16 was confirmed by the ability to produce a rat monoclonal Ab to RhCA 67-16 that showed anti-Id activity for polyclonal rat Ab reactive with AChR residues 67-76. Most importantly, RhCA 67-16 immunization also prevented the development of EAMG in Lewis rats challenged with Torpedo AChR (25% incidence versus 90% in the controls) and diminished the AChR Ab levels in animals injected with low doses of AChR. Our results suggest a therapy for MG and perhaps other autoimmune diseases through the induction of anti-Id Abs by peptide immunogens.

Identification of suppressin-producing cells in the rat pituitary.

Suppressin (SPN) is a novel polypeptide that is synthesized and secreted by normal rat pituitary cells and a rat pituitary tumor cell line, GH3. Specifically, SPN is a negative regulator of growth that inhibits lymphoid and neuroendocrine cell proliferation. The objective of the present study was to identify the cells in the normal rat pituitary that produce SPN. A double immunofluorescence technique, using antibodies to SPN in conjunction with antibodies to the six major adenohypophyseal hormones, was used to colocalize SPN and a specific hormone in a single dispersed pituitary cell. The results of these experiments showed that, on the average, 42% of the cells in the pituitary produce SPN. Suppressin production in the pituitary was restricted to the adenohypophysis. The SPN-producing population in the pituitary was composed of somatotrophs, lactotrophs, corticotrophs, thyrotrophs, and mammosomatotrophs, while gonadotrophs did not produce SPN. Additionally, a PRL reverse hemolytic plaque assay was used to examine SPN production in lactotrophs. The results of these experiments showed that SPN production and the amount of PRL secreted covaried. Specifically, SPN production was observed primarily in non-PRL-secreting lactotrophs or in lactotrophs secreting a high amount of PRL. The results of these experiments suggest a potential regulatory relationship between the synthesis and secretion of SPN and PRL. In summary, this report provides the first identification of SPN-producing cells in the pituitary and shows that SPN production occurs primarily in somatotrophs and lactotrophs.

The structure of a myelin basic protein-associated idiotope.

A cross-reactive idiotope (CRI) has been previously described on monoclonal antibodies (mAbs) specific for encephalitogenic peptides from myelin basic protein (MBP). The anti-CRI mAb, F25F7, binds an idiotope (Id) localized to the light chains of an anti-MBP peptide 1-9 mAb, denoted F23C6, and an anti-MBP peptide 80-89 mAb, denoted 845D3. It is the purpose of this study to further delineate the CRI being recognized by F25F7. To this end, we have found a structural correlation between the CRI and the antigen, a small synthetic peptide, denoted PBM 9-1, used to elicit the anti-Id mAb. Sequence comparison between the light chain of F23C6 and PBM 9-1 reveals a region of homology in CDR 2/FWK 3. The configuration of this site in the VL, as determined by comparison with a mAb, HyHEL-10, whose structure has been determined and is 97% homologous to the light chain of F23C6, conforms to the rules used to define antigenic determinants or Ids. A synthetic peptide having the F23C6 VL CDR 2/FWK 3 sequence inhibited the binding of F25F7 to F23C6 and 845D3. Taken together, these data suggest the Id recognized by F25F7 is defined, in part, by the PBM 9-1-like sequence of F23C6.

Regulation of expression of secretogranin II mRNA in female rat pituitary and hypothalamus.

Secretogranin II (SgII) is an acidic 86-kD protein which is synthesized by most neuroendocrine cells but occurs in greatest abundance in the anterior pituitary gland where it is localized primarily in gonadotrophs. In the present studies, we investigated the regulation of SgII mRNA expression in the anterior pituitary gland by estrogens and gonadotropin-releasing hormone (GnRH) and compared the results to luteinizing hormone beta-subunit (LH beta) mRNA expression. Molecular cloning and nucleotide sequence analysis of a rat pituitary SgII cDNA revealed a derived amino acid sequence identical with that previously reported for the rat adrenal. Not previously reported were five putative nuclear localization signals, four of which coincided with dibasic residues previously thought to serve as proteolytic cleavage sites. In Northern blots, SgII mRNA was found in high abundance in the anterior pituitary gland, in moderate abundance in the brain and adrenal, and in low abundance in the ovary and testis. Measurements of pituitary SgII mRNA during the rat 4-day estrous cycle revealed an inverse relationship with LH beta mRNA: SgII mRNA decreased, whereas LH beta mRNA increased as the cycle progressed. Increases in pituitary SgII mRNA and LH beta mRNA levels occurred after ovariectomy, and decreases occurred after estrogen treatment of such animals. Likewise, pituitary SgII mRNA and LH beta mRNA levels decreased after treatment of ovariectomized animals with a GnRH antagonist. In contrast, ovariectomy significantly decreased SgII mRNA levels in the hypothalamus, and estrogen treatment increased its levels. Our studies reveal that ovarian estrogens and hypothalamic GnRH exert similar effects on SgII mRNA and LH beta mRNA expression in the pituitary. However, since their expression is inverse during the rat estrous cycle, other unidentified regulatory factors with differential effects on their expression may intervene in the regulation of SgII and LH beta mRNA levels.

Thymocytes express a mRNA that is identical to hypothalamic luteinizing hormone-releasing hormone mRNA.

1. A luteinizing hormone-releasing hormone (LHRH)-like molecule produced by thymocytes is similar to hypothalamic LHRH in both bioactivity and antigenicity. 2. We determined whether this thymic LHRH is identical to or only homologous with hypothalamic LHRH by synthesizing and sequencing the cDNA of rat thymus LHRH. 3. The thymocyte and hypothalamic LHRH cDNAs are identical, indicating, that the amino acid sequences of LHRH produced in the hypothalamus and the immune system are also identical. 4. This is the first report showing conclusively that cell of the immune system transcribe the authentic mRNA for a hypothalamic releasing factor, LHRH.

A peptide mimetic of calcium.

Proteins of the troponin superfamily use homologous amino acid sequences as binding sites for Ca2+ and seem to have evolved from an ancestral Ca2+ binding site. We have utilized this ancestral sequence to construct a peptide (Ca(2+)-like peptide) with inverted hydropathy to the calcium-coordinating region of this protein. This synthetic peptide acted like Ca2+ in that (i) it increased the calmodulin-dependent hydrolysis of cAMP by phosphodiesterase, (ii) it interacted with EDTA, and (iii) it enhanced contraction of urinary bladder smooth muscle in vitro. Unlike Ca2+, the peptide's effects were destroyed by acid hydrolysis. These findings demonstrate the synthesis of a peptide that can substitute for Ca2+ and may have considerable utility for the study of Ca(2+)-regulated pathways and possible therapeutic value as a pharmacologic agent.

Detection of growth hormone and growth hormone-releasing hormone-related messenger RNA in rat leukocytes by the polymerase chain reaction.

To validate that growth hormone (GH) and growth hormone-releasing hormone (GHRH) can be produced by leukocytes, we have assessed the presence of GH and GHRH-related mRNA in leukocyte cultures by reverse transcription and the polymerase chain reaction. A sample of the polymerase chain reactions were size-fractionated by electrophoresis in a 0.8% agarose gel and examined with ultraviolet light after ethidium bromide staining. Single major DNA bands corresponding in length to the distance between the 5' ends of the two GH and GHRH specific primers, 603 base pairs and 260 base pairs, respectively, were obtained. The DNA bands hybridized specifically to GH- and GHRH-specific probes after Southern transfer to nitrocellulose. The identity of the GH polymerase chain reaction material was confirmed by restriction enzyme analysis. The results showed that GH and GHRH gene expression occurs in mononuclear leukocytes and support the idea that these neuroendocrine hormones may be common signal molecules between the immune and neuroendocrine systems.

Cloning and direct sequencing from lambda cDNA libraries using the polymerase chain reaction: suppressin and the vasopressin receptor as models.

A strategy using the polymerase chain reaction (PCR) to screen a lambda gt11 pituitary cDNA library for cDNAs encoding suppressin, a putative anti-proliferative protein, and a putative vasopressin receptor is described. The use of this technique will facilitate the demonstration of e.g. the presence of "neuropeptide receptors" on cells of the lymphoid system, confirming the concept of "shared ligands and receptors" by the neuroendocrine and the immune system. Neither of the genes encoding the proteins of the present study have previously been cloned. The PCR-screening procedure requires sequence information from the gene of interest which permits the generation of complementary primers. These primers are then used in combination with lambda phage primers complementary to regions flanking the cloning site in a PCR to amplify cDNAs derived from the gene of interest. This novel screening procedure yields cDNA related to the gene of interest, including the largest clone present in the library. To confirm the utility of this technique for cDNA libraries, the library was also screened using traditional cDNA hybridization techniques. The largest clone obtained by screening the cDNA library with PCR was the same as that obtained by the conventional technique. Thus, the results of these studies show that the PCR method can be used instead of more conventional means to screen cDNA libraries. Lastly, we describe a protocol for directly sequencing PCR-amplified DNA using the same primers that are used for amplification. The combined use of these two strategies permits cloning and sequencing of cDNAs from lambda cDNA libraries in a fraction of the time required using traditional screening techniques, but with identical results.

Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells.

We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl- permeability.

An antisense oligodeoxynucleotide to growth hormone messenger ribonucleic acid inhibits lymphocyte proliferation.

The role of GH in lymphocyte proliferation was studied by examining the effect of an antisense oligodeoxynucleotide (ODN) complementary to GH mRNA. The results of these studies showed that antisense GH ODN treatment inhibits lymphocyte production of immunoreactive GH (irGH). Lymphocytes treated with the GH antisense ODN produced less irGH than did lymphocytes treated with control sense GH ODN. Antisense GH ODN-mediated inhibition of irGH production resulted in a decrease in lymphocyte proliferation. Cells with the antisense GH ODN had less (87%) incorporation of [3H]thymidine [( 3H]TdR) in both resting and Concanavalin-A-stimulated lymphocytes, whereas the incorporation of [3H]TdR in cells treated with a control ODN was not significantly affected. The effect of the antisense ODN on [3H]TdR incorporation was specific, since it could be reversed by hybridization competition with a complementary GH sense ODN or by the addition of exogenous rat GH. Collectively, the data indicate that lymphocytes synthesize and secrete irGH and that irGH produced by these cells can stimulate proliferation, suggesting that GH may play an autocrine/paracrine role in lymphocyte replication.

Corticotropin-releasing factor upregulates expression of two truncated pro-opiomelanocortin transcripts in murine lymphocytes.

Lymphocytes harbor a pro-opiomelanocortin (POMC) mRNA. In this report, a novel procedure was used to study the exonic arrangement of this transcript in lymphocytes. Poly(A)+ mRNA, purified from both corticotropin-releasing factor (CRF)-treated and nontreated lymphocytes, was selectively reverse-transcribed using an antisense oligonucleotide primer complementary to the 3' junction of the translated/nontranslated region of exon 3 of POMC. Alkaline agarose gel analysis of first-strand cDNA synthesis showed an upregulation of POMC transcripts in CRF-treated cells. This first-strand cDNA was amplified in a polymerase chain reaction (PCR) using the complementary antisense primer and selective sense primers homologous to the 5' ends of exons 1, 2, and 3, as well as a region immediately 5' to the ACTH/beta-lipotropin coding region of exon 3 of pituitary POMC. Primers directed at exons 1 and 2 did not amplify a POMC product in nontreated control or CRF-treated cells. However, with both treated and nontreated cells, the internal exon 3 primer amplified the expected size exon 3 DNA fragment (approximately 549 bp). Interestingly, a primer directed at the 5' end of exon 3 apparently did not amplify a POMC product in nontreated cells but did amplify a full-size POMC exon 3 from CRF-treated cells (approximately 615 bp). However, upon reamplification of the original PCR products from nontreated cells, full-length exon 3 product was also observed. Southern gel analysis using a pituitary POMC cDNA probe showed that all of the above PCR products were POMC-related. The results of this study show that lymphocytes basally transcribe at least two POMC transcripts that are upregulated by CRF. These two transcripts lack exons 1 and 2 but contain either part or all of exon 3. The smaller exon 3 transcript was the most abundant transcript under all conditions examined.

Immunomodulatory characteristics of a novel antiproliferative protein, suppressin.

We investigated the immunoregulatory properties of a recently described inhibitor of lymphocyte proliferation, suppressin (SPN). It was determined that preincubation of murine leukocytes with SPN enhances natural killer cell (NK) activity. In addition, SPN potentiates interferon-gamma (IFN-gamma) augmentation of NK activity. Furthermore, preincubation of murine leukocytes with SPN induces the production of IFN-alpha/beta. The IFN-alpha/beta produced is active in NK assays as well as vesicular stomatitis virus neutralization assays. In vivo, SPN increases the time of survival of C57BL/6 mice injected with EL-4 lymphoma cells. Interestingly, SPN inhibits immunoglobulin (IgA, IgG, and IgM) production in response to the mitogen, concanavalin A in a dose-dependent manner. Collectively, the above data indicate SPN may have numerous applications in clinical science including tumor surveillance and autoimmune diseases such as arthritis.

Nucleotide and amino acid sequence of lymphocyte-derived corticotropin: endotoxin induction of a truncated peptide.

To determine the degree of similarity between pituitary and lymphocyte proopiomelanocortin, the lymphocyte mRNA was reverse transcribed, cloned, and sequenced. Murine lymphocyte mRNA was first purified by oligo(dT)-cellulose affinity chromatography and was reverse transcribed by using a selective 3' antisense oligonucleotide primer directed at the boundary between the translated/nontranslated region on the 3' end of exon 3. This cDNA was then amplified in a polymerase chain reaction with selective primers containing Sal I and Kpn I restriction endonuclease sites. Amplified cDNA was then directionally ligated into M13mp18 and M13mp19 bacteriophage and was sequenced. The nucleotide sequence encoding this peptide was identical to that of mouse pituitary corticotropin (ACTH). Elevated levels of lymphocyte immunoreactive ACTH were then induced with bacterial lipopolysaccharide and the peptide(s) was purified by antibody affinity chromatography and reverse-phase high-performance liquid chromatography. The predominant immunoreactive ACTH species was approximately 3 kDa and its sequence was identical to pituitary ACTH(1-25). These results conclusively demonstrate that lymphocytes produce authentic ACTH and harbor its mRNA.