Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

Exogenous quinones inhibit photosynthetic electron transfer in Chloroflexus aurantiacus by specific quenching of the excited bacteriochlorophyll c antenna.

In the photosynthetic green filamentous bacterium Chloroflexus aurantiacus, excitation energy is transferred from a large bacteriochlorophyll (BChl) c antenna via smaller BChl a antennas to the reaction center. The effects of substituted 1,4-naphthoquinones on BChl c and BChl a fluorescence and on flash-induced cytochrome c oxidation were studied in whole cells under aerobic conditions. BChl c fluorescence in a cell suspension with 5.4 microM BChl c was quenched to 50% by addition of 0.6 microM shikonin ((R)-2-(1-hydroxy-4-methyl-3-pentenyl)-5,8-dihydroxy-1, 4-naphthoquinone), 0.9 microM 5-hydroxy-1,4-naphthoquinone, or 4 microM 2-acetyl-3-methyl-1,4-naphthoquinone. Between 25 and 100 times higher quinone concentrations were needed to quench BChl a fluorescence to a similar extent. These quinones also efficiently inhibited flash-induced cytochrome c oxidation when BChl c was excited, but not when BChl a was excited. The quenching of BChl c fluorescence induced by these quinones correlated with the inhibition of flash-induced cytochrome c oxidation. We concluded that the quinones inhibited electron transfer in the reaction center by specifically quenching the excitation energy in the BChl c antenna. Our results provide a model system for studying the redox-dependent antenna quenching in green sulfur bacteria because the antennas in these bacteria inherently exhibit a sensitivity to O(2) similar to the quinone-supplemented cells of Cfx. aurantiacus.     

Mitogenic Activity of Cytoplasmic Membranes Isolated from L-Forms of Staphylococcus aureus

Cytoplasmic membranes of L-forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform-methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform-methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic. The cytoplasmic membranes of the L-forms also exerted a definite mitogenic effect on guinea pig splenocytes, but not on the thymocytes.     

Automated high-performance liquid chromatographic method for determination of furosemide in dog plasma

An automated high-performance liquid chromatographic method for the determination of the diuretic drug furosemide has been established. Dog plasma was injected directly into a two-column system with a BSA—ODS (ODS column coated with bovine serum albumin) precolumn and a C₁₈ analytical column for the separation of furosemide. The two columns were automatically switched. Furosemide remained trapped on the precolumn while proteins were eluted to waste. After column switching, furosemide was washed onto the analytical column and analysed without interference. The greatest advantage of the method is its easy performance without manual sample preparation; it requires no extraction or deproteinization. The method allows determination of 0.1–10 μg/ml of furosemide with accuracy and precision comparable with previously reported values. The coefficients of variation obtained from replicate measurements of 1 μg/ml and 5 μg/ml samples were 1.65% and 2.40%, respectively. This method was used to measure the plasma levels of furosemide in beagle dogs to whom the drugs was administered, as a reference, in a toxicological study.     

Economical Speed and Energetically Optimal Transition Speed Evaluated by Gross and Net Oxygen Cost of Transport at Different Gradients

The oxygen cost of transport per unit distance (CoT; mL·kg^-1·km^-1) shows a U-shaped curve as a function of walking speed (v), which includes a particular walking speed minimizing the CoT, so called economical speed (ES). The CoT-v relationship in running is approximately linear. These distinctive walking and running CoT-v relationships give an intersection between U-shaped and linear CoT relationships, termed the energetically optimal transition speed (EOTS). This study investigated the effects of subtracting the standing oxygen cost for calculating the CoT and its relevant effects on the ES and EOTS at the level and gradient slopes (±5%) in eleven male trained athletes. The percent effects of subtracting the standing oxygen cost (4.8 ± 0.4 mL·kg^-1·min^-1) on the CoT were significantly greater as the walking speed was slower, but it was not significant at faster running speeds over 9.4 km·h^-1. The percent effect was significantly dependent on the gradient (downhill > level > uphill, P < 0.001). The net ES (level 4.09 ± 0.31, uphill 4.22 ± 0.37, and downhill 4.16 ± 0.44 km·h^-1) was approximately 20% slower than the gross ES (level 5.15 ± 0.18, uphill 5.27 ± 0.20, and downhill 5.37 ± 0.22 km·h^-1, P < 0.001). Both net and gross ES were not significantly dependent on the gradient. In contrast, the gross EOTS was slower than the net EOTS at the level (7.49 ± 0.32 vs. 7.63 ± 0.36 km·h^-1, P = 0.003) and downhill gradients (7.78 ± 0.33 vs. 8.01 ± 0.41 km·h^-1, P < 0.001), but not at the uphill gradient (7.55 ± 0.37 vs. 7.63 ± 0.51 km·h^-1, P = 0.080). Note that those percent differences were less than 2.9%. Given these results, a subtraction of the standing oxygen cost should be carefully considered depending on the purpose of each study.     

Nestmate recognition mediated by intestinal bacteria in a termite, Reticulitermes speratus

Discrimination and aggressive responses toward non-nestmates have been observed in a number of termite species, but the mechanism is poorly understood. Here I present the novel hypothesis that differential intestinal bacteria composition leads to production of colony-specific chemical cues that enable nestmate recognition.
The intestinal microflora of a lower termite, Reticulitermes speratus , consisted of many bacteria species. The composition of the intestinal bacteria was exclusively colony-specific. Termites that had adsorbed an unfamiliar odor of bacteria sampled from another colony were fiercely attacked by nestmates. Experimental manipulation of the composition of bacteria by antibiotics successfully changed the recognition behavior toward nestmates. These results indicate that intestinal bacteria play an important role in nestmate recognition.     

Effect of Osmotic Stress on Turgor Pressure in Mung Bean Root Cells

Turgor pressure in cells of the elongating region of intactmung bean roots was directly measured by using the pressure-probetechnique. After the external osmotic pressure had been increasedfrom 0 MPa to 0.5 MPa, turgor pressure rapidly decreased byabout 0.5 MPa from 0.65 MPa to 0.14 MPa and root elongationstopped. Subsequent turgor regulation was clearly confirmed,which followed the osmotic adjustment to maintain a constantdifference in the osmotic pressure between root-cell sap andthe external medium ( II). It took at least 6 h for turgor pressureto recover to an adjusted constant level of about 0.5 MPa dueto turgor regulation, but rootelongation resumed within onlyan hour after the osmotic treatment. Therefore, the resumptionof root elongation under osmotic stress could not have beendirectly connected with turgor regulation. Furthermore, sincethe amounts of decrease in turgor pressure just after applicationsof various degrees of osmotic stress could be interpreted inrelation to those in II, hydraulic conductivity between theinside and the outside of root cells must be large enough toattain water potential equilibrium rapidly in response to osmoticstress. We conclude that turgor pressure in the cells of theelongating region of mung bean roots is determined mainly by II because of water potential equilibrium. (Received January 27, 1987; Accepted May 21, 1987)     

Electron flow and heme-heme interaction between cytochromes b-558, b-595 and d in a terminal oxidase of Escherichia coli

The ESR signals of the cytochromes in the Escherichia coli terminal oxidase cytochrome d complex were studied at cryogenic temperature. The intensities and g values of the rhombic high-spin signals changed when the electronic state of cytochrome d was changed from the oxidized state to the reduced or oxygen-binding or CO-binding state. These rhombic signals were therefore assigned to cytochrome b-595, which is located near cytochrome d in the oxidase complex. This assignment was supported by the finding that the Em value of the rhombic signals differed from that of cytochrome d (Hata, A. et al. (1985) Biochim. Biophys. Acta 810, 62-72). Photolysis and ligand-exchange experiments with the reduced CO complex of the oxidase were performed in the presence of oxygen at -140 degrees C. The ESR spectra of three intermediate forms trapped by controlled low temperatures were detected. These forms were designated as the oxygen-binding intermediate I (ESR-silent), oxygen-binding intermediate II (giving ESR signals at g = 6.3, 5.5 and 2.15), and oxygen-binding intermediate III (giving signals at g = 6.3, 5.5 and 6.0). From these results, electron flow in the cytochrome d complex is proposed to proceed in the order, cytochrome b-558----cytochrome b-595----cytochrome d----O2. A model of the mechanism of four-electron chemistry for oxidation of ubiquinol-8 and formation of H2O by the cytochrome d complex is presented.