A new regulatory element that augments the Tax-dependent enhancer of human T-cell leukemia virus type 1 and cloning of cDNAs encoding its binding proteins.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) trans activates the 21-bp enhancer of HTLV-1. A sequence of more than two copies of the 21-bp enhancer is efficiently activated by Tax, but one copy is not activated extensively. Another sequence (TRE-2, positions -163 to -117) adjacent to the 21-bp enhancer in the long terminal repeat of HTLV-1 can enhance a single copy of the 21-bp enhancer activity in trans activation by Tax. This sequence contains motifs related to the Ets- and NF-kappa B-binding sequences, but mutations at these sites indicated that neither is responsive to cooperation with the 21-bp enhancer. A deletion mutation of TRE-2 identified 25 bases at positions -158 to -134 (TRE-2S) as an essential sequence, and TRE-2S was sufficient to give maximum cooperation with one copy of the 21-bp enhancer in trans activation by Tax protein. Using TRE-2S as a probe, we screened a cDNA library of HUT102 cells by the Southwestern (DNA-protein) procedure and isolated two cDNA clones, THP-1 and -2. These two clones encode TRE-2S-binding proteins, and they differ by only an extra 17 amino acids in THP-2. Both THP proteins contain five zinc finger motifs which are strikingly similar to those of the GLI family, an amplified gene product in glyoma cells. The binding site of THP-1 and -2 was GAACCACCCA in TRE-2S, which is highly homologous to the GLI-binding site. These results suggest that binding of THP to TRE-2S may be involved in cooperation with one copy of the 21-bp enhancer in responding to Tax trans activation.     

An Improved Enrichment Broth for Isolation of Escherichia coli O157, with Specific Reference to Starved Cells, from Radish Sprouts

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.     

Purification and characterization of rat dopamine beta-monooxygenase and monoclonal antibodies to the enzyme

Dopamine beta-monooxygenase was extensively purified from rat adrenal. The specific activity of the final preparation was approx. 1500 nmol/min per mg protein, which was much higher than the highest yet reported. As judged by gel filtration on Ultrogel AcA22, SDS-polyacrylamide gel electrophoresis, and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 88 000. The isoelectric point of the enzyme was estimated to be pH 6.6 in the presence of 8 M urea. Spleen cells from BALB/c mice immunized with rat dopamine beta-monooxygenase were fused to P3-X63-Ag8-653 mouse myeloma cells. From 55 hybrid cells, 10 stable clones secreting anti-dopamine beta-monooxygenase antibody were obtained. Antibody from one clone was coupled to CNBr-activated Sepharose 4B and the monoclonal antibody-Sepharose was shown to be very useful to isolate rat dopamine beta-monooxygenase from crude preparations.     

Studies on the generation of Ca2+/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme.

The mechanism for the generation of the Ca2+/calmodulin (CaM)-independent activity of calmodulin-dependent protein kinase II (CaM-kinase II) by autophosphorylation was studied by characterizing the autothiophosphorylated enzyme, which is resistant to hydrolysis. When CaM-kinase II was incubated with adenosine 5'-O-(thiotriphosphate) at 5 degrees C, the incorporation of thiophosphate into the enzyme occurred rapidly, reaching a maximum level within a few minutes, in parallel with increase in Ca2+/CaM-independent activity. The maximum level was 1 mol of thiophosphate per mol of subunit of the enzyme, and the thiophosphorylation occurred exclusively at Thr286 in the alpha subunit and Thr287 in the other subunits of the enzyme. These results, taken together, indicate that the autothiophosphorylation of Thr286/Thr287 of each subunit is involved in the generation of the Ca2+/CaM-independent activity. The activity of the autothiophosphorylated enzyme, when assayed in the presence of Ca2+/CaM, showed the same kinetic properties as did the Ca2+/CaM-dependent activity of the original non-phosphorylated enzyme, but when assayed in the absence of Ca2+/CaM, it showed the same Vmax as the Ca2+/CaM-dependent activity but higher Km values for protein substrates. Thus, the phosphorylation of Thr286/Thr287 of the subunit of the enzyme by autophosphorylation appears to not only enhance the affinity of its substrate-binding site for the protein substrate, although it is lower than that of the enzyme activated by the binding of CaM, but also convert the active site to the fully active state.     

Interstitial stem cell proliferation in hydra: evidence for strain-specific regulatory signals.

We have examined the growth behavior of small numbers of interstitial stem cells transplanted into tissue of genetically unrelated strains of Hydra magnipapillata. We show that such stem cells, which are at low density following transplantation, proliferate more rapidly than the stem cells of the host, which are at normal density. The rapid proliferation is similar to the proliferation rate of stem cells transplanted into interstitial cell free tissue. The results suggest that stem cells transplanted into heterotypic tissue are unable to "sense" the presence of host stem cells and to adopt their growth rate to that of the surrounding cells. Thus, the feedback signal which negatively regulates stem cell growth as a function of stem cell density must be strain specific.     

Conversion of Tyrosine Hydroxylase to Stable and Inactive Form by the End Products

We have reported previously that tyrosine hydroxylase in the crude extract from rat striatum exists in the inactive form showing almost no activity at the physiological pH and that the inactive form is produced by the action of the end products of the enzyme, such as dopamine. The incubation of the enzyme with the end products resulted in not only the inactivation but also a remarkable stabilization of the enzyme. Catechols possessing amino groups but no negatively charged groups on the side chains (catecholamine-type catechols) were effective at a concentration as low as 10(-7) M in both the inactivation and stabilization of the enzyme. In contrast, catechols not possessing positively or negatively charged side chains (3,4-dihydroxyphenylethyleneglycol-type catechols) were ineffective at a concentration of 10(-7) M but effective at a concentration of 10(-6) M for both the inactivation and stabilization. Catechols possessing negatively charged groups (3,4-dihydroxyphenylacetic acid-type catechols) were ineffective even at a concentration of 10(-6) M. Thus, the end products of tyrosine hydroxylase appear to serve to keep the enzyme inactive and stable. The reaction mechanism of the conversion of the enzyme from the active/labile form to the inactive/stable form by dopamine was also investigated.     

Taxonomic study of the Lactobacillus acidophilus group, with recognition of Lactobacillus gallinarum sp. nov. and Lactobacillus johnsonii sp. nov. and synonymy of Lactobacillus acidophilus group A3 (Johnson et al. 1980) with the type strain of Lactobacillus amylovorus (Nakamura 1981).

Biochemical properties and DNA-DNA reassociation studies of Lactobacillus acidophilus strains isolated from humans and animals indicate that these include six genomospecies. Two new species can be differentiated from the established species of the genus Lactobacillus: L. gallinarum sp. nov. (type strain, ATCC 33199) and L. johnsonii sp. nov. (type strain, ATCC 33200). Furthermore, it was clarified that L. acidophilus group A3 (Johnson et al. 1980) is synonymous with L. amylovorus.     

Hepatitis B virus envelope L protein particles. Synthesis and assembly in Saccharomyces cerevisiae, purification and characterization.

The hepatitis B virus envelope gene encodes three transmembrane proteins in frame; S, the product of S gene; M, the product of M (pre-S2 + S) gene; and L, the product of L (pre-S1 + pre-S2 + S) gene. Unlike the S and M proteins, attempts to efficiently synthesize L proteins and assemble them into L protein particles in various eukaryotic cells have been unsuccessful, probably because of the presence of the pre-S1 peptide with an unknown function which appears to be inhibitory to the host secretory apparatus. To investigate the role of the pre-S1 peptide, we constructed an L gene fused with a synthetic gene for chicken-lysozyme signal peptide (C-SIG) at the 5'-terminal and placed the resultant gene under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. After the fused-C-SIG peptide was correctly processed by the yeast secretory apparatus, a yeast transformant synthesized a protein with a molecular mass of approximately 52 kDa at a level of 42% of the total soluble protein. Electron micrographic observation showed that the gene products assembled into 23-nm spherical and filamentous particles. The pre-S peptide of the gene product was deposited into the endoplasmic reticulum (ER) lumen and well-glycosylated. It seemed that the gene products were accumulated as particles in certain specific membrane structures of the yeast secretory apparatus. Moreover, both the amount of mRNAs specific for the L gene and the in vivo stability of the synthesized L proteins did not change significantly by the addition of the C-SIG gene. These findings indicated that, if the pre-S1 peptide penetrates the ER membrane efficiently, the L proteins can be synthesized cotranslationally, translocate across the ER membrane with its S region, and then assemble by themselves into the particle form. Therefore, the pre-S1 peptide may involve weak or reduced signal peptide activity for recognition by the secretory apparatus and/or for the transport of the pre-S peptide into the ER lumen.     

Homeostatic recovery of interstitial cell populations in Hydra.

The growth of interstitial cell populations in Hydra magnipapillata was examined following transplantation of small numbers of interstitial cells into "epithelial animals" which lacked all cell types in the interstitial cell lineage. The distribution pattern of transplanted interstitial cells during the growth phase was examined by staining whole animals with toluidine blue and cell numbers were determined by maceration. The following results were obtained: (1) Transplanted interstitial cells formed a contiguous patch which spread distoproximally but not circumferentially. (2) The displacement of interstitial cells from parents to buds was a random process; buds incorporated interstitial cells only when they were formed in the vicinity of the patch. (3) Interstitial cells increased exponentially in number with a doubling time of 1.8 days for at least 10 days after transplantation, which is faster than the normal doubling time of 2.8 days. (4) The self-renewal probability at low interstitial cell levels was estimated to be 0.72, which was higher than the normal value of 0.64. This increase was attained by lowering the fraction of nematocyte differentiation. These results indicate that the homeostatic recovery of interstitial cell populations is attained by increasing the self-renewal probability rather than by preferential retention of interstitial cells in parent animals at the expense of buds (Heimfeld, 1985).     

Regulation of ascorbate oxidase expression in pumpkin by auxin and copper

Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation.