Robert D. LeBoeuf

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 ∼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 inDrosophila, 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.

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.

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.

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-α and-β 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. Additional evidence supporting this hypothesis was provided by experiments showing that by blocking endogenous SPN activity in vitro with neutralizing anti-SPN antibodies unstimulated lymphocyte proliferation is induced. Lastly, SPN also inhibits the proliferation of leukemia and lymphoma cells in vitro, suggesting a potential role for SPN in regulating tumor cell proliferation.

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.

Possible alternate splicing or initiation of the pro-opiomelanocortin gene in lymphocytes

Des lymphocytes en culture transcrivent le gene de la proopiomelanocortine; cette transcription augmente quand les lymphocytes sont cultives en presence de ACTH-RH

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 ex‐pression 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.

[20] – Purification and Characterization of Immunoregulatory Peptides from Neuroendocrine Tissues: Suppressin as a Model

Publisher Summary This chapter describes the purification and characterization of immunoregulatory peptides from neuroendocrine tissues using suppressin as a model. It has been proposed that bidirectional communication between the immune and neuroendocrine systems occurs molecularly as a result of a sharing of receptors and ligands. The same molecules can be used for intrasystem regulation and intersystem communication. It has been predicted that tissues of neuroendocrine origin would be a fertile source for the discovery of new immunoregulatory proteins and that the immune system would contain new hormones. This chapter discusses studies that are consistent with this idea. It discusses the purification and characterization of a novel immunoregulatory molecule of pituitary origin termed suppressin. When using an activity as a means of purification, it is essential to rapidly establish that the substance is not a previously identified molecule. The chapter discusses the development of a monospecific polyclonal antibody to suppressin and using this to screen a pituitary cDNA library. The sequence of the positive clone then clearly establishes the uniqueness of the factor.