Induction in mouse peritoneal macrophages of 34 kDa stress protein and heme oxygenase by sulfhydryl-reactive agents

The synthesis of 34-kDa stress protein was enhanced, with a simultaneous increase in heme oxygenase activity, when mouse macrophages were exposed to diethylmaleate or sodium arsenite. After 7 h of exposure to the sulfhydryl agents, the 34-kDa protein was the most actively synthesized protein. Immunoblot analysis showed that the induced 34-kDa protein reacted with an antibody raised against bovine heme oxygenase. Cadmium ions or 1-chloro-2,4-dinitrobenzene also induced the 34-kDa protein which reacted with the antibody. Treatments of the cells with buthionine sulfoximine or hydrogen peroxide weakly induced the protein, while diamide treatment or heat shock was without effect. These results are consistent with our previous findings that heavy metal ions including arsenite and cadmium ions induce heme oxygenase (32-kDa stress protein) in human cell lines [Taketani, S., Kohno, H., Yoshinaga, T., & Tokunaga, R. (1989) FEBS Lett. 245, 173-176], and also suggest that the formation of glutathione conjugate with sulfhydryl-reactive agents may mediate the induction of the stress protein in mouse peritoneal macrophages.     

Fourier transform infrared spectroscopic study on retinochrome and its primary photoproduct, lumiretinochrome

Structural studies of retinochrome, and its photoproduct, lumiretinochrome, were done by Fourier transform infrared difference spectroscopy. The absorption bands in the carbonyl stretching region which shift in D2O show the changes in the protein part during the photoreaction. Strong absorption bands in the finger-print region show that the all-trans-retinal chromophore in retinochrome isomerizes to the 11-cis-retinal chromophore in lumiretinochrome upon illumination with yellow-green light at 83K.     

Molecular cloning of cDNA for proteasomes from rat liver: primary structure of component C3 with a possible tyrosine phosphorylation site

Proteasomes are multicatalytic proteinase complexes consisting of multiple components. Previously, we reported the cloning and sequencing of cDNA for the largest component, C2, of rat liver proteasomes [Fujiwara, T., Tanaka, K., Kumatori, A., Shin, S., Yoshimura, T., Ichihara, A., Tokunaga, F., Aruga, R., Iwanaga, S., Kakizuka, A., & Nakanishi, S. (1989) Biochemistry 28, 7332-7340]. In the present study, the nucleotide sequence of another component (C3) of proteasomes has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The deduced sequence of component C3 consists of 234 amino acid residues with a calculated molecular weight of 25,925. These values are consistent with those obtained by protein chemical analyses. A single mRNA species hybridizing to the C3 cDNA of rat liver was expressed in all rat tissues examined and in a variety of other eukaryotic organisms, its distribution being similar to that of C2 mRNA. The wide distribution of the gene product, possibly C3, suggests that this protein functions similarly in most eukaryotes. C3 is an unmodified protein of a single gene and differs from any other known protein, but its overall amino acid sequence resembles those of other proteasomal components, including C2, suggesting that these components belong to a single family of proteins with the same evolutionary origin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of the hemopexin receptor from human placenta

A hemopexin receptor detected in detergent-solubilized placental membranes was purified from the human placenta, using hemopexin-Sepharose affinity chromatography. The solubilized membranes exhibited binding sites of 2.77 pmol of hemopexin/mg of protein with a dissociation constant (Kd) of 6.6 X 10(-8) M. The purified receptor has a molecular weight of 80,000, determined on sodium dodecyl sulfate-gel electrophoresis. Immunoinhibition experiments using the antibody against the placental receptor revealed inhibition of binding of 125I-hemopexin to human leukemia K562 and HL 60 cells, thereby strongly supporting that the polypeptide isolated from the human placenta was the hemopexin receptor.     

Mapping molar shapes on signaling pathways

A major challenge in evolutionary developmental biology is to understand how genetic mutations underlie phenotypic changes. In principle, selective pressures on the phenotype screen the gene pool of the population. Teeth are an excellent model for understanding evolutionary changes in the genotype-phenotype relationship since they exist throughout vertebrates. Genetically modified mice (mutants) with abnormalities in teeth have been used to explore tooth development. The relationship between signaling pathways and molar shape, however, remains elusive due to the high intrinsic complexity of tooth crowns. This hampers our understanding of the extent to which developmental factors explored in mutants explain developmental and phenotypic variation in natural species that represent the consequence of natural selection. Here we combine a novel morphometric method with two kinds of data mining techniques to extract data sets from the three-dimensional surface models of lower first molars: i) machine learning to maximize classification accuracy of 22 mutants, and ii) phylogenetic signal for 31 Murinae species. Major shape variation among mutants is explained by the number of cusps and cusp distribution on a tooth crown. The distribution of mutant mice in morphospace suggests a nonlinear relationship between the signaling pathways and molar shape variation. Comparative analysis of mutants and wild murines reveals that mutant variation overlaps naturally occurring diversity, including more ancestral and derived morphologies. However, taxa with transverse lophs are not fully covered by mutant variation, suggesting experimentally unexplored developmental factors in the evolutionary radiation of Murines.     

A novel yeast secretion vector utilizing secretion signal of killer toxin encoded on the yeast linear DNA plasmid pGKL1

The NH2-terminal signal region comprising of approximately 70% length of the prepro-sequence of the pGKL killer precursor protein was found to direct an efficient secretion of the mouse alpha-amylase into the culture medium of Saccharomyces cerevisiae. The alpha-amylase molecule secreted into the culture medium was identified by both immuno-blotting and assay of the enzyme activity. The amount of alpha-amylase secreted via the killer toxin signal was comparable to that directed by the leader sequence of mating factor alpha. The secretion of alpha-amylase using the killer toxin signal was blocked at 37C but not at 25C in sec18-1 host, indicating that alpha-amylase is exported through the normal secretion pathway of S. cerevisiae.     

A monoclonal antibody with broad reactivity to human interferon-alpha subtypes useful for purification of leukocyte-derived interferon

A monoclonal antibody to human interferon-alpha, termed HT-1 antibody, with a broad reactivity to various subtypes of interferon-alpha was prepared. It bound and neutralized all of the four subtypes of E. coli-derived human recombinant interferon-alpha (alpha 1, alpha 2, alpha 4, and alpha 6) tested; it also neutralized human natural leukocyte interferon but only partially. Human interferon-beta and -gamma were not bound. The antibody conjugated to Sepharose beads retained over 90% of human leukocyte interferon induced by Sendai virus. The bound interferon was recovered by acid elution in good yields and in almost pure form (specific activity was about 2 X 10(8) international units/mg protein). The purified interferon showed, in SDS-polyacrylamide gel electrophoresis, an activity profile with major peaks in a mol. wt. range of 17,000-22,000, which completely agreed with the profile shown by polyclonal antibody-purified interferon. Such purified leukocyte interferon-alpha preparations containing most of the naturally occurring subtypes can be useful for clinical and other purposes.     

Direct measure of fluorescence intensity for efficient receptor-binding assay: conjugates of ethinylcarboxyestradiol and 5(and 6)-carboxyfluorescein via alpha, omega-diaminoalkanes as a tracer for estrogen receptor

Steroidal nuclear receptors (NRs) have been acknowledged as a target binding protein of so-called endocrine disruptors. It is therefore necessary to develop an efficient assay system for screening these endocrine-disrupting chemicals. We here describe the first exemplification of a direct measure of fluorescence intensity for a binding assay of NRs. We designed and synthesized a series of conjugates of 17alpha-ethinylcarboxyestradiol with carboxyfluorescein, both carboxyl groups of which were cross-linked with alpha,omega-diaminoalkanes. The resulting fluorescein-linked estradiol derivatives E2(n)cF (n=2, 4, 6, 8, 10 and 12) were evaluated for their fluorescence and receptor-binding characteristics. E2(4)cF and E2(8)cF exhibited the sufficient binding affinity to the recombinant estrogen receptor (ER) in the radiolabel binding assay using [(3)H]17beta-estradiol, and showed excellent fluorescent characteristics in the fluorescence measurements with and without ER. They exhibited sufficiently large specific binding characteristics with adequate K(d)- and B(max)-values. When these fluorescent ligands were used as a tracer for the binding assay against the ER, assay data of various compounds were shown to be compatible with those obtained from the ordinary binding assay using [(3)H]17beta-estradiol. The present study clearly shows that measurement of fluorescence intensity, instead of fluorescence polarization, affords an adequate receptor-binding assay system.     

Crucial role of peroxiredoxin III in placental antioxidant defense of mice

We observed frequent stillbirth in peroxiredoxin III (PrxIII) knockout maternal mice. Quantitative real time PCR (qRT-PCR) and Western-blot analysis revealed increased oxidative stress in placentas that were deficient in PrxIII. We did not find significant difference between PrxIII knockout maternal mice and wild-type littermates in hematological parameters, fetal number, and embryonic development. Nevertheless, we noticed enhanced expression of PrxI in erythrocytes of pregnant knockout mice. Our results provided in vivo evidence that PrxIII played a crucial role in placental antioxidant defense. Up-regulation of PrxI might provide a compensation that protected erythrocytes against oxidative damage.