Sequence-specific DNA recognition by basic peptides.

A chiral template with C2 symmetry has been used for modeling a dimeric interface of DNA binding protein. An oligopeptide derived from the basic region of MyoD, a recently described "helix-loop-helix" class of DNA binding protein, has been tethered to the template. Among the four models which differ in chirality and polarity with respect to the arrangement of two subunits, only one dimer model with right-handed and C-terminus to C-terminus arrangement of the peptide subunits binds DNA containing native MyoD binding sequence.     

The origin of the luteinizing hormone-releasing hormone (LHRH) neurons in newts (Cynops pyrrhogaster): the effect of olfactory placode ablation

Summary Neurons containing luteinizing hormone-releasing hormone (LHRH) are first detected in newt embryos (Cynops pyrrhogaster) in the olfactory epithelium and ventromedial portion of the olfactory nerve, after which they sequentially appear in the intracerebral course of the terminal nerve at prometamorphosis, and in the septo-preoptic area at postmetamorphosis. In adults, however, LHRH-immunoreactive cells are rarely seen in the nasal region, and their distribution shifts into the brain, suggesting their migration. In order to ascertain the origin and possible migration route of these neurons in newt larvae, the effect of unilateral or bilateral olfactory placodectomy on the LHRH neuronal system has been studied. Removal of the olfactory placode results in the absence of LHRH-immunoreactive cells in the nasal and brain regions of the operated side, whereas the subsequent growth and the LHRH-immunoreactive cellular distribution in the contralateral side are identical to those of normal larvae. Following bilateral placodectomy, no LHRH immunoreactivity is detected on either side of the olfactory-brain axis. These results suggest that LHRH neurons of the newt, Cynops pyrrhogaster, originate in the olfactory placode and then migrate into the brain during embryonic development.     

Generation of 19 STS markers that can be anchored at specific sites on human chromosome 21.

Sequence-tagged sites (STSs) are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR) and can be used to construct long-range physical maps of chromosomal DNA. These STSs can be detected by PCR assays developed by reference to data obtained from the sequencing of restriction fragment length polymorphism-DNA markers for chromosome 21, which were derived from recombinant lamba-phage and plasmid clones made from DNA of a human-hamster hybrid cell line. In this report, we describe the generation of 19 new STSs that are specific for human chromosome 21.     

The promotive effect of interleukin 4 with interleukin 2 in the proliferation of tumor-infiltrating lymphocytes from patients with malignant tumor

In adoptive immunotherapy, the number of effector cells is one of the major factors relating to the therapeutic efficacy. We demonstrated that tumor-infiltrating lymphocytes (TILs) were stimulated to proliferate by incubation with interleukin 2 (IL-2) plus interleukin 4 (IL-4). TILs cultured with IL-2 plus IL-4 increased 3.1-fold more than TILs cultured with IL-2 alone. However, IL-4 did not alter the cytotoxic activity of TILs against autologous tumor cells and established tumor cell lines. It is suggested that IL-2 receptor is related to the mechanism of the proliferation of activated TILs cultured by combination with IL-2 and IL-4. Thus, the combination of IL-2 and IL-4 may increase the efficacy of adoptive immunotherapy using activated TILs.     

Effect of Supra-High Irradiation on the Photosynthetic System of the Cyanophyte Synechocystis PCC 6714

Stability of thylakoid components under supra-high irradiancewas studied with the cyanophyte Synechocystis PCC 6714. Theactivity of overall photosynthesis was quickly inactivated (T_1/2=20min) under supra-high irradiance (300 W m^–2, white light).In parallel with the inactivation of photosynthesis, QA in PSII was also inactivated. Both inactivations were acceleratedby chloramphenicol (CAP) addition. The reactivation of PS IIrequired weak irradiation and was suppressed by CAP. However,PS I measured as P700 was very stable. The level of PS I measuredas P700 was not significantly reduced by the irradiation for12 h even in the presence of CAP while the level of Cyt b₅₅₉,component of PS II, was decreased markedly. The function ofPS I before and after supra-high irradiation with CAP was examinedby comparing sizes of P700 oxidation induced by a short flash,by a continuous light, and by determination of O₂-and ferredoxin-reduction.No difference was observed in PS I actions before and afterthe irradiation treatment. These results indicate that the PSI complex is very tolerant of supra-high irradiation. However,the cells grown under supra-high irradiance contained much fewerPS I and PS II complexes than Cyt b₆–f complexes. Theformer levels were reduced to a half to one fourth of thosebefore growth while the level of Cyt b₆–f complex wasnot reduced so much. A possible mechanism for changes in thylakoidcomposition under supra-high irradiation was discussed. (Received February 16, 1991; Accepted June 12, 1991)     

Porcine brain natriuretic peptide-like immunoreactivity in rat tissues

The presence of immunoreactive porcine brain natriuretic peptide in rat tissues was studied with a specific radioimmunoassay for porcine brain natriuretic peptide-26. The cross-reactivity of the antiserum used was less than 0.001% with rat atrial natriuretic peptide, rat brain natriuretic peptide-32 and rat brain natriuretic peptide-45. Immunoreactive porcine brain natriuretic peptide was detectable in various tissues of the rat, and high concentrations of immunoreactive porcine brain natriuretic peptide were found in the brain and cardiac atrium, with the highest level in the hypothalamus (159±30 fmol/gram wet tissue, mean±SEM, n=4). Reverse phase high performance liquid chromatography showed that the immunoreactive porcine brain natriuretic peptide of the whole brain and heart extracts eluted mainly at an identical position to synthetic porcine brain natriuretic peptide-26. These findings indicate that porcine brain natriuretic peptide-like substance, distinct from rat brain natriuretic peptide, is present in high concentrations in the rat brain and cardiac atrium.     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.     

Cell type-specific expression of the human renin gene.

We have previously produced transgenic mice carrying the human renin gene, whose expression is regulated in a tissue-specific manner. In the present study, we further characterized expression of the transgene. Northern blot analysis showed that the human renin gene is expressed in the kidney but not in the liver of two lines of transgenic mice with 10 and 50 copies of the transgene, suggesting that the integrated copy number of the human renin gene does not influence the dominant-renal expression pattern. Immunohistochemical study using a monoclonal antibody specific for human renin demonstrated that expression of human renin in the transgenic mouse kidney is confined to the epithelioid juxtaglomerular cells. Transfection experiments indicated that the chloramphenicol acetyltransferase fusion gene containing the 3-kb upstream sequences of the renin gene is activated only in human epithelioid embryonic 293 cells derived from kidney but not in human HepG2 cells from liver. These findings suggest that transfer of the cloned renin gene into mice and in vitro cultured cell lines can give rise to cell type-specific expression.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.