Kinetics of hydrogen-deuterium exchange in ATPase from a thermophilic bacterium PS3.

The kinetics of the hydrogen-deuterium exchange reaction in a stable ATPase (TF₁) from a thermophilic bacterium PS3 was followed by infrared absorption measurements. The rates of the hydrogen-deuterium exchange reactions decreased in following order; free form, TF₁·ADP, TF₁·ATP and TF₁·AMP-P(NH)P. TF₁ does not dissociate into subunits even in the absence of nucleotides, thus differences in exchange likely reflect differences in conformations of subunits. These results indicate that the structure is most restricted when ATP or AMP-P(NH)P is bound to the enzyme.     

Fluorescence fading and stabilization in cytofluorometry

The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as "33258 Hoechst" and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO2 and cresyl-violet-SO2, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromolecule-dye complexes generally induce fluorescence stabilization.     

Purification and preparation of antibody to RNA polymerase II stimulatory factors from Ehrlich ascites tumor cells.

An improved method was developed for purification of the protein termed S-II that specifically stimulates RNA polymerase II of Ehrlich ascites tumor cells. The specific activity of the final preparation was 400 000 units/mg of protein, which is about 30-fold higher than that of the previous preparation [Sekimizu, K., et al. (1976) Biochemistry 15, 5064]. The final preparation gave a single band on both sodium dodecyl sulfate and nondenaturing gel electrophoresis, and the protein extracted from the band on nondenaturing gel had stimulatory activity. S-II is a basic protein with a molecular weight of 40 500. The fundamental characteristics of S-II determined with the previous preparation were confirmed with completely purified S-II. A specific antibody to S-II was prepared. This antibody inhibited only the stimulatory activity of S-II and did not affect the activity of RNA polymerase II itself. Thus, S-II is probably not a component of the multimeric proteins of RNA polymerase II.     

Distribution of the messenger RNA coding for the common precursor of corticotropin and beta-lipotropin within the bovine pituitary.

The distribution of the mRNA coding for the common precursor of corticotropin and beta-lipotropin among different parts of the bovine pituitary has been investigated by quantifying the mRNA activity with the use of a cell-free protein-synthesizing system. The results obtained have demonstrated that this mRNA activity is located both in the anterior lobe and in the intermediate lobe, while it is essentially not detectable in the neural lobe nor in the stalk. The structural identity of the translation products of corticotropin/beta-lipotropin mRNA from the anterior and from the intermediate lobe has been indicated by their molecular weight as well as by the electrophoretic patterns of the peptide fragments formed from them upon partial enzymatic proteolysis or upon cyanogen bromide cleavage. The specific activity of corticotropin/beta-lipotropin mRNA in the intermediate lobe is about 20-fold higher than that in the anterior lobe, and the total activity of this mRNA in the former is about 2-fold higher than that in the latter. In the intermediate lobe, the translation product of corticotropin/beta-lipotropin mRNA amounts to almost one-third of the products encoded by total translatable mRNA. These results indicate that corticotropin/beta-lipotropin mRNA represents a major mRNA species in intermediate lobe of the pituitary, thus suggesting that this lobe may perform a highly specialized function in producing a large amount of the common precursor of corticotropin and beta-lipotropin.     

Serum surfactant protein D is increased in acute and chronic inflammation in mice

Surfactant protein A (SP-A) and surfactant protein D (SP-D) are important components of innate immunity that can modify the inflammatory response. However, alterations and regulation of SP-A and SP-D in acute and chronic inflammation are not well defined. In addition, serum SP-D may serve as a biomarker of lung inflammation. We determined the expression of SP-A and SP-D in murine models. To study acute inflammation, we instilled bleomycin intrabronchially. To study chronic lung inflammation, we used a transgenic mouse that overexpresses tumor necrosis factor (TNF)-alpha under the control of the SP-C promoter. These mice have a chronic mononuclear cell infiltration, airspace enlargement, pulmonary hypertension, and focal pulmonary fibrosis. In acute inflammation model, levels of mRNA for all surfactant proteins were reduced after bleomycin administration. However, serum SP-D was increased from days 7 to 28 after instillation. In chronic inflammation model, SP-D mRNA expression was increased, whereas the expression of SP-A, SP-B and SP-C was reduced. Both serum and lung SP-D concentrations were increased in chronic lung inflammation. These data clarified profile of SP-A and SP-D in acute and chronic inflammation and indicated that serum SP-D can serve as a biomarker of lung inflammation in both acute and chronic lung injury in mice.     

In vivo reopening of the neonatal ductus arteriosus by a prostanoid EP4-receptor agonist in the rat

Prostaglandin E1 is used to reopen the constricted ductus arteriosus in neonates with ductus-dependent circulation. To clarify possible prostanoid receptor agonists that can reopen the neonatal ductus with fewer side effects, we studied in vivo reopening of the neonatal ductus arteriosus by AE1-329, a prostanoid EP4-receptor agonist, in the rat. Neonatal rats were incubated at 33 degrees C. The inner diameter of the ductus was measured with a microscope and a micrometer following rapid whole-body freezing. Intraesophageal pressure was measured with a Millar micro-tip transducer. The ductus arteriosus constricted quickly after birth, and the inner diameter was 0.80 and 0.08 mm at 0 and 60 min after birth. PGE1 and AE1-329, injected subcutaneously at 60 min after birth, dilated the ductus dose-dependently. Thirty minutes after injection of 10 ng/g of PGE1 and AE1-329, the ductus diameter was 0.52 and 0.65 mm, respectively. The ductus-dilating effect of PGE1 was maximal at 15-30 min, and disappeared at 2 h. The ductus-dilating effect of AE1-329 was prolonged, the ductus was widely open at 6 h, and closed at 12 h after injection of 10 ng/g AE1-329. AE1-259-01 (EP2 agonist) (100 ng/g) did not dilate the neonatal ductus. Respiration was depressed by PGE1, but not by AE1-329. These results indicate the major role of EP4 in the neonatal ductus and that AE1-329, an EP4 agonist, can be used to dilate the neonatal constricted ductus without the side effects shown by EP3, including apnea.     

Chromosomal assignment and deletion mapping of barley EST markers

From about 10000 PCR-based EST markers of barley we chose 1421 EST markers that were demonstrated to be amplified differently by PCR between wheat (Triticum aestivum cv. Chinese Spring) and barley (Hordeum vulgare cv. Betzes). We assigned them to the seven barley chromosomes (1H to 7H) by PCR analysis using a set of wheat-barley chromosome addition lines. We successfully assigned 701 (49.3%) EST markers to the barley chromosomes: 75 to 1H, 127 to 2H, 119 to 3H, 94 to 4H, 108 to 5H, 81 to 6H and 97 to 7H. By using a set of Betzes barley telosomic addition lines of Chinese Spring, we could successfully determine the chromosome-arm (S or L) location of at least 90% of the EST markers assigned to each barley chromosome. We conducted a trial mapping using 90 EST markers assigned to 7HS (49) or 7HL (41) and 19 wheat lines carrying 7H structural changes. More EST markers were found in the distal region than in the proximal region.     

The production-to-respiration ratio and its implication in Lake Biwa,Japan

Production-to-respiration (P:R) ratio was estimated at an offshore site of Lake Biwa in order to examine whether the plankton and benthic community is subsidized with allochthonous organic carbon, and to clarify the role of this lake as potential source or sink of carbon dioxide. The respiration rate of protozoan and metazoan plankton was calculated from their biomass and empirical equations of oxygen consumption rates, and that of bacterioplankton was derived from their production rate and growth efficiency. In addition, the carbon mineralization rate in the lake sediments was estimated from the accumulation rate of organic carbon, which was determined using a ²¹⁰Pb dating technique. On an annual basis, the sum of respiration rates of heterotrophic plankton was comparable to net primary production rate measured by the ¹³C method. However, when the mineralization rate in the lake sediments was included, the areal P:R ratio was 0.89, suggesting that Lake Biwa is net heterotrophic at the offshore site with the community being subsidized with allochthonous organic carbon. Such a view was supported by the surface water pCO₂ that was on average higher than that of the atmosphere. However, the estimate of net CO₂ release rate was close to that of carbon burial rate in the sediments. The result suggests that the role of Lake Biwa in relation to atmospheric carbon is almost null at the offshore site, although the community is supported partially by organic carbon released from the surrounding areas.