cis-Elements and Protein Factors Related to Regulation of Transcription of Arylsulfatase Gene during Sea Urchin Development

Eight restriction fragments (I–VIII) were prepared to cover a whole span of the enhancer region in the upstream of the Ars gene of the sea urchin, Hemicentrotus pulcherrimus , and their abilities to influence on the Ars gene expression were estimated by CAT assay. Only three fragments (III, IV and V) encompassing a 0.6 kb region between −2.8 kb and −2.2 kb stimulated CAT expression. By mobility shift assays, it was found that the Ars enhancer region is composed of multiple cis -acting elements that interact with nuclear proteins in a sequence-specific manner. Among them, two sequences, a G-string and a GATCTCCCC, were determined by DNA footprinting as sites of protein-DNA interaction. The DNA-binding factor prevalence changed ontogenically in three different patterns. Possible activation of DNA-binding proteins through their modification is discussed.     

Relationship between cardiac output and oxygen uptake at the onset of exercise

The purpose of the present study was to assess the relationship between the rapidity of increased gas exchange (i.e. oxygen uptake ) and increased cardiac output ( ) during the transient phase following the onset of exercise. Five healthy male subjects performed multiple rest-exercise or light exercise (25 W)-exercise transitions on an electrically braked ergometer at exercise intensities of 50, 75, or 100 W for 6 min, respectively. Each transition was performed at least eight times for each load in random order. The was obtained by a breath-by-breath method, and was measured by an impedance method during normal breathing, using an ensemble average. On transitions from rest to exercise, rapidly increased during phase I with time constants of 6.8–7.3 s. The also showed a similar rapid increment with time constants of 6.0–6.8 s with an apparent increase in stroke volume (SV). In this phase I, increased to about 29.7%–34.1% of the steady-state value and increased to about 58.3%–87.0%. Thereafter, some 20 s after the onset of exercise a mono-exponential increase to steady-state occurred both in and with time constants of 26.7–32.3 and 23.7–34.4 s, respectively. The insignificant difference between and time constants in phase I and the abrupt increase in both and SV at the onset of exercise from rest provided further evidence for a cardiodynamic contribution to following the onset of exercise from rest.     

Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.     

Efficient and gentle elution of antibodies from an immobilized polypeptide antigen BT saturated magnesium chloride

Summary A model antigen, rabbit immunoglobulin G, was immobilized onto polyester cloth by adsorption. The antigen cloth was reacted with sheep anti-rabbit IgG antibody. Antibody bound to the antigen cloth was nearly quantitatively eluted by saturated MgCl₂, whereas a commercial antibody eluent slowly eluted only about 70 % of the antibody. Exposure of antibody to saturated MgCl₂ for 30 min resulted in no loss of immunoactivity. Saturated MgCl₂, therefore, is an ideal eluent in immunoaffinity purification of antibodies.     

Tissue-Specific Isoforms of Catalase Subunits in Castor Bean Seedlings

Catalases purified from endosperm glyoxysomes and non-specializedmicrobodies from hypocotyls of castor bean seedlings differedin their specific activity [90–164 and 0.89–4.9kunits (mg protein)^–1, respectively] and in their constituentsubunits [two subunits of 54 and 56 kDa for the endosperm enzymeand only one of 56 kDa for the hypocotyl enzyme]. Immunoblotanalysis also showed that particulate fractions from the endospermsand from etiolated and green cotyledons contained two catalasesubunits of 54 and 56 kDa, whereas such fractions from the hypocotylsand roots contained only the 56-kDa subunit. Leaf peroxisomesfrom green leaves had two catalase subunits of around 55 kDaeach. Results of translation in vitro indicated that the 54-and 56-kDa subunits were translated from distinct mRNAs andlevels of both mRNAs increased in the endosperms during germination,prior to increases in levels of catalase proteins. In the hypocotyls,the 56-kDa subunit seemed to be synthesized constitutively. ¹Present addresses: YO, Toyota Central Institute, 31-9 Musashizuka,Nagabuchi, Nagakute, Aichi 480-11, Japan     

Phototropism in hypocotyls of radish. VI. No exchange of endogenous indole-3-acetic acid between peripheral and central cell layers during first and second positive phototropic curvatures

Using a physicochemical method, the distribution of endogenous indole-3-acetic acid (IAA) was measured in the peripheral and central cell layers, as well as in the illuminated and shaded sides of phototropically stimulated radish hypocotyls (Raphanus sativus var. hortensis f. gigantissimus Makino). The IAA was evenly distributed over the illuminated and shaded sides in the first and second positive phototropic curvatures induced by a pulse or a continuous unilateral illumination with blue light. Also, no net exchange of the IAA between the peripheral and central cell layers was observed during these curvatures. These results suggest that phototropism of radish hypocotyls cannot be described by the Cholodny-Went theory.     

Nuclear localization activity of phytochrome B

Phytochromes are soluble red/far-red-light photoreceptor proteins which mediate various photomorphogenic responses of plants. Despite much effort, the signal transduction mechanism of phytochrome has remained obscure. Phytochromes are encoded by a small multigene family in Arabidopsis . Among the members of the family, phytochrome A (phyA) and B (phyB) are the best characterized. PhyB contains putative nuclear localization signals within its C-terminal region. Transgenic Arabidopsis plants were produced which expressed a fusion protein consisting of GUS and C-terminal fragments of phyB. GUS staining from the fusion protein in these transgenic plants was observed in the nucleus, which suggests that the nuclear localization signal of the fragment is functional. Next, it was examined whether the endogenous phyB was detected in the nucleus. Nuclei were isolated from the light-grown wild-type Arabidopsis leaves and subjected to the immunoblot analysis. The result indicated that a substantial fraction of total phyB was recovered in the isolated nuclei. This result was further confirmed by the immunocytochemical analysis of the protoplasts. Finally, the effects of light treatments on the levels of phyB in the isolated nuclei were examined. Dark adaptation of the plants before the nuclear isolation reduced the levels of phyB. The reduction was accelerated by irradiation of plants with far-red light before the transfer to darkness. Thus, nuclear localization of phyB was suggested to be light-dependent.     

Distribution of various nickel compounds in rat organs after oral administration

In this study, eight kinds of nickel (Ni) compounds were orally administered to Wistar male rats and the distribution of each compound was investigated 24 h after the administration. The Ni compounds used in this experiment were nickel metal [Ni−M], nickel oxide (green) [NiO(G)], nickel oxide (black) [NiO(B)], nickel subsulfide [Ni₃S₂], nickel sulfide [NiS], nickel sulfate [NiSO₄], nickel chloride [NiCl₂], and nickel nitrate [Ni(NO₃)₂]. The solubilities of the nickel compounds in saline solution were in the following order; [Ni(NO₃)₂>NiCl₂>NiSO₄]≫[NiS>Ni₃S₂]>[NiO(B)>Ni−M>NiO(G)]. The Ni level in the visceral organs was higher in the rats given soluble Ni compounds; Ni(NO₃)₂, NiCl₂, NiSO₄, than that in the rats receiving other compounds. In the rats to which soluble Ni compounds were administered, 80–90% of the recovered Ni amounts in the examined organs was detected in the kidneys. On the other hand, the Ni concentration in organs administered scarcely soluble Ni compounds; NiO(B), NiO(G), and Ni−M were very low. The estimated absorbed fraction of each Ni compounds was increased with the increase of the solubility. These results suggest that the kinetic behavior of Ni compounds administered orally is closely related with the solubility of Ni compounds, and that the solubility of Ni compounds is one of the important factors for determining the health effect of Ni compounds.     

Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Halotolerant Green Alga Dunaliella tertiolecta

A Ca²⁺-dependent protein kinase (CDPK) that has been partiallypurified and characterized previously [Yuasa and Muto (1992)Arch. Biochem. Biophys. 296: 175] was further purified to about20,000-fold from the soluble fraction of Dunaliella tertiolecta.The enzyme preparation contained 60- and 52-kDa polypeptidesboth of which phosphorylated casein as a substrate. Both polypeptidesshowed a Ca²⁺-dependent increase in mobility during SDS-PAGEand ⁴⁵Ca²⁺-binding activity after SDS-PAGE and electroblottingonto a nitrocellulose membrane, suggesting that both the 60-and 52-kDa CDPKs directly bind Ca²⁺. The protein kinase inhibitors,K-252a and staurosporine, inhibited the CDPK competitively withrespect to ATP. An antibody raised against the 60-kDa CDPK crossreactedwith both the 60- and 52-kDa polypeptides. Both molecular specieswere autophosphorylated in the presence of Ca²⁺, and a highlyphosphorylated 80-kDa band appeared in addition to these phosphorylatedbands at 60 and 52 kDa in SDS-PAGE. However, the specific activityof CDPK was not changed by prior autophosphorylation when theautophosphorylated enzyme was assayed as a mixture of thesephosphorylated molecular species. Only the 60-kDa polypeptidewas immunodetected in subcellular fractions of Dunaliella cells.The 52-kDa polypeptide increased during storage of the enzyme.These results suggest that the 52-kDa polypeptide is a proteolyticartifact produced during purification. Immunoreactive bandsof 60-kDa were detected in extracts of several green algae butnot in extracts of higher plants or a brown alga. ¹This research was partly supported by Grants-in-Aid from theMinistry of Education, Science and Culture, Japan (No. 06454013and 06304023) and Research Fellowship of the Japan Society forthe Promotion of Science for Young Sciencists. ²Research Fellow (PD) of the Japan Society for the Promotionof Science.