Characterization of human thyrotropin receptor-related peptide-like immunoreactivity in peripheral blood of Graves' disease.

An antiserum raised against an alignment of amino acid-(32-56), termed TSHRP-1, in the extracellular domain of human thyrotropin (TSH) receptor was used to identify the TSH receptor-like substance in plasma of Graves' disease. The dilution curve of plasma TSHRP-1-like immunoreactivity was observed in a manner parallel to the standard synthetic peptide curve in radioimmunoassay, and its molecular weight estimated approximately 60 kDa. The amounts of TSHRP-1-like immunoreactivity were significantly higher in Graves' plasma than those in plasma of normal and hypothyroid patients due to Hashimoto's thyroiditis. The present results indicate that human peripheral blood possesses a soluble form of the extracellular domain of TSH receptor which may contribute to the pathophysiology of Graves' disease.     

A novel anti-HIV synthetic peptide, T-22 ([Tyr5,12,Lys7]-polyphemusin II).

Tachyplesin and polyphemusin are antimicrobial peptides recently isolated from the hemocytes of horseshoe crabs (Tachypleus tridentatus and Limulus polyphemus). We synthesized them and their analogs and examined their antiviral activity against human immunodeficiency virus (HIV) type 1 in vitro. The infection of human T cells with the virus was markedly inhibited by some of them at low concentrations. In this structure-activity study, we found that [Tyr5,12, Lys7]-polyphemusin II, which was designated as T22, had extremely high anti-HIV activity. Its 50% inhibitory concentration (EC50) was 0.008 micrograms/ml, while its 50% cytotoxic concentration (CC50) was 54 micrograms/ml and these values were comparable to those of AZT. This result indicates that T22 would be a potential candidate for the therapy of HIV infection.     

Differential regulation of thyrotropin-releasing hormone receptor mRNA levels by thyroid hormone in vivo and in vitro (GH3 cells).

We studied the effect of thyroid status on thyrotropin-releasing hormone receptor (TRH-R) mRNA levels both in vivo and in vitro (GH3 cells) using a cloned rat TRH-R cDNA by RT-PCR. Experimental hypothyroid rats were produced by total thyroidectomy and were then killed 7 days after the operation. TRH receptor binding in the anterior pituitary and serum TSH level were elevated approximately 2-fold and 8-fold, respectively, in 7 day thyroidectomized rats. TRH-R mRNA levels in hypothyroid rats were also increased significantly compared with those of normal rats. In GH3 cells, however, no significant change of TRH-R mRNA level was observed between cultures treated with triiodothyronine (T3, 10(-9) and 10(-7) M) and the untreated group. The present data indicate that 1) the in vivo effects of thyroid status on TRH-R mRNA levels differ from the in vitro one, and that 2) the down regulation of TRH-R binding by thyroid hormone in GH3 cells may be mediated by translational or post-translational mechanisms.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

Long-Term Acceptance of Major Histocompatibility Complex-Mismatched Cardiac Allograft Induced by a Low Dose of CTLA4IgM Plus FK506

The immunosuppressant FK506 prolongs allograft survival. However, at therapeutic doses it has significant side effects. A fusion protein consisting of the extracellular portion of CTLA4 and the Fc portion of human IgG (CTLA4IgG) also prolongs allograft survival, but large doses of CTLA4IgG are required for the induction of cardiac allograft acceptance. Therefore, we constructed a pentameric form of a new CTLA4 fusion protein, CTLA4IgM. We tested whether low doses of CTLA4IgG or CTLA4IgM in combination with subtherapeutic doses of FK506 can prolong allograft survival in a synergistic fashion. C57BL/6 (H-2^b) neonatal hearts were transplanted to CBA/J (H-2^k) mice in a heterotopic, nonvascularized cardiac allograft model. The findings demonstrate that a combination of low doses of FK506 plus a pentameric form of CTLA4Ig, CTLA4IgM, leads to significant graft survival, while a combination of FK506 plus CTLA4IgG does not.     

Effect of penicillin G on the electroporation of Rhodococcus rhodochrous CF222

M. SUNAIRI, N. IWABUCHI, K. MURAKAMI, F. WATANABE, Y. OGAWA, H. MUROOKA AND M. NAKAJIMA. 1996. Suitable conditions for the introduction of bacteriophage DNA into cells of Rhodococcus rhodochrous CF222 by electroporation were established, and penicillin G was found to enhance the transfection frequency. When conditions optimal for the parental strain were applied to its colony-morphological mutants, different transfection frequencies were observed. Penicillin G enhanced the transfection frequency of smooth and mucoidal mutants but not of rough mutants.     

Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)