H-2RIIBP expressed from a baculovirus vector binds to multiple hormone response elements.

H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex class I genes. Based on its homology with Drosophila XR2C/CF1, H-2RIIBP may play a role in development. By using a baculovirus expression system, a large amount of recombinant H-2RIIBP was produced. The recombinant protein accumulated in the nucleus of insect cells. A series of monoclonal antibodies reacting with the recombinant H-2RIIBP was then generated. A DNA-protein immunoprecipitation assay was developed with these antibodies, enabling the DNA-binding specificity of H-2RIIBP to be distinguished from that of an endogenous region II binding factor expressed in uninfected insect cells. We show that H-2RIIBP binds to estrogen response elements with an affinity comparable to that for the region II enhancer. H-2RIIBP also bound to some, but not all, thyroid hormone response elements and retinoic acid response elements, albeit at a lower affinity. Binding to these elements was demonstrated without exogenous addition of a ligand. The H-2RIIBP binding specificity determined by this assay was in agreement with the specificity assessed by Southwestern and gel mobility shift assays. Furthermore, methylation interference assays indicated that H-2RIIBP recognizes the conserved hormone response motif GG(T/A)CA. Taken together, these data demonstrate that H-2RIIBP is capable of binding to hormone response elements of a variety of genes. They suggest that H-2RIIBP may exert a pleiotropic function.     

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.     

Detection of H-2K mRNA in mouse 8-cell embryo by cDNA cloning

Mouse MHC class I gene expression in 8-cell embryo was examined by cDNA cloning. We constructed a cDNA library from 8-cell embryos of ICR mice and isolated a class I cDNA from 3.0 x 10(5) phage clones of the library. Sequencing analysis of this clone revealed it to include the cDNA fragment extending from the exon 6 of the cytoplasmic portion to 3' untranslated region 1 of H-2K gene. Qa, Tla or other embryonic class I cDNA have not been isolated in the library.     

Insulin-degrading enzyme is capable of degrading receptor-bound insulin

In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ agglutinin column and monoclonal anti-IDE antibody affinity column, respectively. [125I]insulin-receptor complex was incubated with various amounts of IDE at 0 degree C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135 kd band (insulin receptor alpha-subunit) decreased, whereas that of 110 kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37 degrees C, about half of the [125I]insulin was dissociated from the complex. However, the time course of [125I]insulin degradation in this incubation was essentially identical to that of free [125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [125I]insulin from insulin-receptor complex incubated with IDE was significantly (p less than 0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

Stimulation of Amino Acid Uptake and Na+, K+-ATPase Activity by Norepinephrine in Superior Cervical Sympathetic Ganglia Excised from Adult Rats

Active uptake of a labelled nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), into isolated superior cervical sympathetic ganglia (SCG) excised from adult rats was considerably stimulated by the addition of either norepinephrine (NE, 50 microM) or 3,4-dihydroxyphenylethylamine (dopamine, DA, 100 microM) to the medium during aerobic incubation for 2 h at 37 degrees C. The NE-induced increase in AIB uptake was significantly antagonized by the addition of an alpha 1-adrenoceptor antagonist (prazosin, 10 microM) in SCG axotomized 1 week prior to the examination, in which most of the ganglionic neurons had degenerated and reactive proliferation of the satellite glial components was in progress. The addition of neither acetylcholine (ACh, 1 mM) plus eserine (0.1 mM) nor cyclic nucleotides (1 mM) changed the AIB uptake by the SCG. In the axotomized SCG, the NE-evoked increase in AIB uptake was much more pronounced than that of intact or denervated SCG. A kinetic study of the active AIB uptake in the SCG showed that NE produced a decrease of the Km value and an increase in the Vmax, especially in the axotomized SCG. Ganglionic Na+, K+-ATPase activity was greatly stimulated in the presence of NE, but not by ACh. These results strongly suggest that the NE-induced enhancement of active AIB uptake in the isolated SCG is occurring in glial cells rather than in neuronal cells, with a possible alteration of membrane properties for amino acid uptake and with an apparent regulation by the stimulated transport enzyme Na+, K+-ATPase.     

Simulation analysis of excitation conduction in the heart: Propagation of excitation in different tissues

The normal excitation and conduction in the heart are maintained by the coordination between the dynamics of ionic conductance of each cell and the electrical coupling between cells. To examine functional roles of these two factors, we proposed a spatially-discrete model of conduction of excitation in which the individual cells were assumed isopotential. This approximation was reasoned by comparing the apparent space constant with the measured junctional resistance between myocardial cells. We used the four reconstruction models previously reported for five kinds of myocardial cells. Coupling coefficients between adjacent cells were determined quantitatively from the apparent space constants. We first investigated to what extent the pacemaker activity of the sinoatrial node depends on the number and the coupling coefficient of its cells, by using a one-dimensional model system composed of the sinoatrial node cells and the atrial cells. Extensive computer simulation revealed the following two conditions for the pacemaker activity of the sinoatrial node. The number of the sinoatrial node cells and their coupling coefficients must be large enough to provide the atrium with the sufficient electric current flow. The number of the sinoatrial node cells must be large so that the period of the compound system is close to the intrinsic period of the sinoatrial node cell. In this simulation the same sinoatrial node cells produced action potentials of different shapes depending on where they were located in the sinoatrial node. Therefore it seems premature to classify the myocardial cells only from their waveforms obtained by electrical recordings in the compound tissue. Second, we investigated the very slow conduction in the atrioventricular node compared to, for example, the ventricle. This was assumed to be due to the inherent property of the membrane dynamics of the atrioventricular node cell, or to the small value of the coupling coefficient (weak intercellular coupling), or to the electrical load imposed on the atrioventricular node by the Purkinje fibers, because the relatively small atrioventricular node must provide the Purkinje fibers with sufficient electric current flow. Relative contributions of these three factors to the slow conduction were evaluated using the model system composed of only the atrioventricular cells or that composed of the atrioventricular and Purkinje cells. We found that the weak coupling has the strongest effect. In the model system composed of the atrioventricular cells, the propagation failure was not observed even for very small values of the coupling coefficient.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts

Using affinity chromatography with immobilized gelatin and native type I collagen, we have identified the major collagen-binding proteins in Nonidet P-40 extracts of chick embryo fibroblasts labeled with [35S] methionine. After washing the gelatin- or collagen-Sepharose beads with high ionic strength buffer, a 47,000-dalton protein was the only major protein besides fibronectin found to bind to these affinity beads. The isoelectric point of this protein was approximately 9.0, with a closely spaced minor spot. The total amount and the synthesis of this collagen-binding protein were both decreased in Rous sarcoma virus-transformed cells. This collagen-binding protein was found to be phosphorylated by incubating intact cells with [32P]orthophosphate. Phosphoamino acid analysis revealed that serine and threonine residues were phosphorylated, but tyrosine was not. Although quantities of the 47,000-dalton protein labeled with [35S]methionine were decreased by a factor 2.5 after transformation, the incorporation of [32P]orthophosphate/unit of protein was 5-7-fold higher in transformed cells. In temperature-sensitive mutant virus-infected cells, the amount of the 47,000-dalton protein was also decreased at the temperature permissive for transformation, and the incorporation of [32P]orthophosphate/protein was also increased. These studies establish that a major membrane-associated collagen-binding protein of fibroblasts is phosphorylated and that it is altered in both total quantity and degree of phosphorylation after malignant transformation.