In vitro packaging and replication of individual genomic segments of bacteriophage phi 6 RNA.

The genome of bacteriophage phi 6 contains three segments of double-stranded RNA. Procapsid structures whose formation was directed by cDNA copies of the large genomic segment are capable of packaging the three viral message sense RNAs in the presence of ATP. Addition of UTP, CTP, and GTP results in the synthesis of minus strands to form double-stranded RNA. In this report, we show that procapsids are capable of taking up any of the three plus-strand single-stranded RNA segments independently of the others. In manganese-containing buffers, synthesis of the corresponding minus strand takes place. In magnesium-containing buffers, individual message sense viral RNA segments were packaged, but minus-strand replication did not take place unless all three viral single-stranded RNA segments were packaged. Since the conditions of packaging in magnesium buffer more closely resemble those in vivo, these results indicated that there is no specific order or dependence in packaging and that replication is regulated so that it does not begin until all segments are in place.     

Acid growth effects in maize roots: Evidence for a link between auxin-economy and proton extrusion in the control of root growth

The role of proton excretion in the growth of apical segments of maize roots has been examined. Growth is stimulated by acidic buffers and inhibited by neutral buffers. Organic buffers such as 2[N-morpholino] ethane sulphonic acid (MES) — 2-amino-2-(hydroxymethyl)propane-1,3 diol (Tris) are more effective than phosphate buffers in inhibiting growth. Fusicoccin(FC)-induced growth is also inhibited by neutral buffers. The antiauxins 4-chlorophenoxyisobutyric acid (PCIB) and 2-(naphthylmethylthio) propionic acid (NMSP) promote growth and H⁺-excretion over short time periods; this growth is also inhibited by neutral buffers. We conclude that growth of maize roots requires proton extrusion and that regulation of root growth by indol-3yl-acetic acid (IAA) may be mediated by control of this proton extrusion.Abbreviations IAA indol-3yl-acetic acid - ABA abscisic acid - FC fusicoccin - PCIB 4-chlorophenoxy-isobutyric acid - MES 2(N-morpholino)ethane sulphonic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3-diol - NMSP 2-(naphthylmethylthio)propionic acid     

Inhibition of shoot geotropism by neutral buffers

Submerged hypocotyl sections from Helianthus have been used to test the effect of neutral buffers on shoot geotropism. When hypocotyls have been abraded, it is found that increasing the molarity (0.25 to 20 mm) of pH 6.8 K-phosphate buffer, as well as other buffering systems, results in a strong inhibition of geotropic curvature. Buffer strength has no such effect on the curvature of nonabraded segments. One possible explanation for these data is that asymmetric shoot growth following geostimulation may require the establishment of a proton gradient across the cell walls of the shoot. When neutral buffers have access to the wall space (i.e. in abraded segments), they may prevent the establishment of such a gradient.     

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.     

Experimental alterations of neurofilaments and neurotubules by calcium and other ions

Short segments of desheathed rat peripheral nerve were immersed in phosphate, tris-HCl and cacodylate buffers containing varying concentrations and admixtures of CaCl₂, AgCl, MgCl₂, PbCl₂, and AlCl₃ before fixation. These tissues were subsequently processed for electron microscopic examination.     

Oxalate transport across the isolated rat colon. A re-examination

A net absorption of oxalate and chloride was observed when isolated, shortcircuited segments of rat colon were bathed by a calcium-containing buffer. Removal of calcium promoted a two-fold decrease in transmural resistance, while the net chloride flux was reduced and the net oxalate transport abolished. It was concluded that net oxalate absorption was not observed in previous studies (employing calcium-free buffers) because calcium is required to maintain the integrity of the conductive pathways across colonic epithelia.     

A shared buffer architecture for interactive VOD servers

Video-on-demand (VOD) servers need to be efficiently designed in order to support a large number of users viewing the same or different videos at different rates. While considering a disk-array based VOD server, use of a shared buffer at the server end may be more economical than the sole use of dedicated buffers at each user's end. In this paper, we propose a simple buffer sharing architecture that may be used when disk-array based video servers are used. Our aim is to support the maximum number of users for a given number of video server disks while employing a simple scheme requiring less buffer space. The number of video segment retrievals that can occur within a certain time (the service round) is maximum when the scan disk scheduling algorithm is used. Consequently, we shall assume use of the scan algorithm for disk retrieval. The VOD server has a buffer manager that directs retrieved segments to appropriate buffer locations depending on their release and deadlines. The release and deadlines of segments are such that buffer requirement at the user's set-top box is minimized to two video segments while avoiding video starvation and buffer overflow at the user's end. We propose a novel scheme for the operation of the shared buffer that aims at increasing buffer utilization and decreasing cell loss due to buffer overflow. An ATM based broadband network is assumed and all segments are stored in buffers as fixed length ATM cells. We also use a novel scheme for grouping frames into segments and illustrate its advantages over earlier ones. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of ferrous sulfate and pH on L-dopa absorption

Ferrous sulfate decreases L-dopa bioavailability in humans probably as a result of binding of L-dopa by iron in the gastrointestinal tract. This study was conducted to determine if iron by binding L-dopa decreases L-dopa absorption and to investigate the effect of different pH buffers on intestinal absorption of L-dopa in the presence and absence of ferrous sulfate. A rat model developed to examine drug absorption was used. Control animals had buffered [14C]L-dopa solutions injected into two in vivo closed segments of intestine; a 5-cm duodenal and a 5-cm proximal jejunal segment. These studies were conducted using solutions buffered at pH 5.5, 6.5, 7.5, and 8.5. An identical procedure was followed for experimental animals except ferrous sulfate was injected with the buffered L-dopa solutions. Ferrous sulfate resulted in a reduction in L-dopa absorption in the buffers at all pHs in both the duodenum and jejunum. The average reduction in L-dopa absorption in the presence of iron was 22.6% in the duodenum and 23.9% in the jejunum. There was a tendency for ferrous sulfate to cause a greater reduction in L-dopa absorption as the buffer pH increased. There was also a decrease in L-dopa absorption in the higher pH buffers in the absence of iron. Despite this latter result, in the jejunum there was an increase in the percent reduction in L-dopa absorption associated with ferrous sulfate as pH increased. Although this tendency was not as consistent in the duodenum as the jejunum, the combined results are compatible with the chemical model of increased L-dopa--iron binding as pH increases.     

The “acid growth effect” and geotropism

Summary The curvature developed by segments of sunflower hypocotyl exposed to gravitational stimulus was enhanced in buffer solutions between pH 3.4 and 4.0 in the absence of added auxin. This effect was observed both when the segments were submerged during the stimulus and when they floated near the surface of the solution. 5–10 min in a horizontal position was sufficient to induce subsequent curvature.Straight growth of the segments was also promoted in buffers of this pH range.The acid effect on curvature was insensitive to KAsO₂, HgCl₂ and cycloheximide, inhibitors which drastically reduced auxin-induced curvature. Furthermore, acid buffer, but not auxin, restored the ability of segments taken from etiolated and starved plants to respond to gravity. These results suggest that the polarisation following gravistimulus may not be resticted to the asymmetric distribution of auxin and auxin co-factors but may involve a general physiological asymmetry.     

Proteolytic modifications of the carboxyl-terminal region of H-2Kk

Conditions were established for the generation of limited proteolysis products from purified H-2Kk in high yield (greater than 70%). Chymotrypsin, trypsin, or papain treatment in buffer containing Nonidet P-40 resulted in removal of discrete segments from the H-2 heavy chain without detectable alteration of the beta 2-microglobulin. The Mr = 47,400 heavy chain was converted to products with Mr = 44,200, 42,800, or 40,600 by treatment with chymotrypsin, trypsin, or papain, respectively. Papain digestion removed both the hydrophilic carboxyl terminus and the hydrophobic regions. The size, detergent binding properties, and products resulting from subsequent papain treatment demonstrated that chymotrypsin or trypsin removed segments of the hydrophilic carboxyl-terminal region of the heavy chain while leaving the hydrophobic (membrane-spanning) and glycosylated NH2-terminal regions intact. Chymotrypsin and trypsin caused rapid and extensive degradation of the H-2Kk heavy chain when treatment was done in buffer containing deoxycholate, suggesting that the protein undergoes partial, but readily reversible, denaturation in this detergent. This may account for the elution of H-2K and D antigens from monoclonal antibody affinity columns by deoxycholate-containing buffers.     

An Improved Method for Detecting Auxin-induced Hydrogen Ion Efflux from Corn Coleoptile Segments

Conditions necessary to detect maximal auxin-induced H⁺ secretion using a macroelectrode have been investigated using corn coleoptile segments. Auxin-induced H⁺ secretion is strongly dependent upon oxygenation or aeration when the tissue to volume ratio is high. Cuticle disruption or removal is also necessary to detect substantial auxin-induced H⁺ secretion. The auxin-induced decrease in pH of the external medium is stronger when the hormone is applied to tissue in which the cuticle has been disrupted with an abrasive than when the hormone is applied to tissue from which the cuticle and epidermis have been removed by peeling. The lower detectable acidification of the external medium when using peeled segments appears to be due in part to the leakage of buffers into the medium and in part to the removal of the auxin-sensitive epidermal cells.     

Infectious diarrhea of infant rats produced by a rotavirus-like agent.

During the investigation of an outbreak of diarrhea in suckling rats, a virus morphologically identical to but antigenically distinct from rotaviruses was identified. The disease was characterized clinically by erythema and cracking and bleeding of the perianal skin associated with the excretion of poorly formed fecal pellets, liquid, and gas. Light microscopy-observable changes consisted of small intestinal villous atrophy, villous epithelial necrosis, and villous epithelial syncytial cell formation. The cytoplasm of the epithelial syncytial cells contained large numbers of 80-nm viral particles that were often associated with reticular aggregates of electron-dense material. Viral infection principally involved the luminal one-fourth to one-third of the intestinal villi as determined by indirect immunofluorescence. This rotavirus-like agent contained 11 double-stranded RNA segments; however, the migration pattern of these segments in polyacrylamide gels differed from the electrophoretic pattern which is characteristic of the typical rotaviruses. The agent had a buoyant density in CsCl of 1.36 to 1.4 g/cm3 and was labile at pH 3 and at 56 degrees C; however, infectivity of viral inocula was not altered by extensive treatment with ether or by pH 5 buffers. This disease, which we have named infectious diarrhea of infant rats, is the first recognized viral diarrhea of rats and appears to be a good model for the study of the recently recognized group of atypical rotaviruses.     

pH-Dependence of Extension Growth in Avena Coleoptiles and Its Implications for the Mechanism of Auxin Action

The pH-dependence of acid-induced growth in excised segments of Avena sativa coleoptiles has been reinvestigated in the pH range 3 to 7. In contrast to previous reports (e.g. DL Rayle [1973] Planta 114: 63-73), only acidic buffers with a pH below 5.0 induce an extension response. A pH of 3.5 to 4.0 is required to mimic auxin-mediated growth. Very similar pH-response curves are obtained with both intact (abraded) and peeled coleoptiles. These results agree with the recent finding of a similarly low sensitivity to protons in maize coleoptiles. It is shown that the apparently much higher sensitivity to protons previously reported for peeled Avena coleoptiles is due to incubating the tissue in buffer of pH 6.8 between peeling and measuring the effect of acidic buffers. Neutral pH reversibly inhibits the spontaneous extension burst originating on release from tissue tension after removing the epidermis. Reversal of this inhibition can be achieved by buffers of pH 5.0 to 6.0 (or distilled water), thereby simulating an acid-induced growth response in this pH range. It is concluded that true acid-induced wall-loosening generally does not take place above pH 5.0 and that a pH considerably below 4.0 is required in order to stimulate growth to an extent comparable to that obtained in response to auxin. The “acid-growth theory,” which requires an acid-mediated loosening of the cell wall in the pH range 5 to 6, this pH being established by auxin-induced proton excretion, can therefore also not be substantiated in Avena.     

Effect of Xyloglucan Oligosaccharides on Growth,Viscoelastic Properties,and Long-Term Extension of Pea Shoots

The growth-promoting effect of xyloglucan-derived oligosaccharides was investigated using a bioassay with entire pea (Pisum sativum L., var Alaska) shoots. After a 24-h incubation period at 25[deg]C, xyloglucan oligosaccharide (XGO) solutions with concentrations of 10-6 M notably increased the growth rate of pea shoots, whereas the same oligosaccharides at 10-7 M were less effective. To investigate the possible correlation between growth rate changes in the XGO-treated shoots and changes in the wall mechanical properties of their growing regions (third internodes), we used a short-term creep assay. The promotion of elongation by XGOs was reflected in an enhancement of the viscoelasticity of the growing regions of the shoots. To show whether this effect on wall viscoelastic properties was the cause or a consequence of their growth promotion, we tested the effect of XGOs on the long-term extension of isolated cell walls. We characterized an acid-induced extension in isolated cell walls from pea shoots that was not inhibited by preincubation in neutral buffers. Exogenously added XGOs did not alter the pattern of pea segment extension at any pH tested, indicating that XGOs have no direct effect on cell wall viscoelasticity. Finally, preincubation of pea segments in neutral buffers with XGOs enhanced their capacity to extend under acidic conditions. This finding suggests that XGOs at a neutral pH can act via transglycosylation, weakening the wall matrix and making the wall more responsive to other mechanisms of acid-induced extension as an expansin-mediated extension.     

Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride

Enthalpy and heat capacity changes for the deprotonation of 18 buffers were calorimetrically determined in 0.1 M potassium chloride at temperatures ranging from 5 to 45°C. The values of the dissociation constant were also determined by means of potentiometric titration. The enthalpy changes for the deprotonation of buffers, except for the phosphate and glycerol 2-phosphate buffers, were found to be characterized by a linear function of temperature. The enthalpy changes for the second dissociation of phosphate and glycerol 2-phosphate where divalent anion is formed on dissociation were fitted with the second order function of temperature rather than the first order. Temperature dependence of buffer pH calculated by using the enthalpy and heat capacity changes obtained was in good agreement with the temperature variation of the pH values actually measured in the temperature range between 0 and 50°C for all the buffers studied. On the basis of the results obtained, a numeric table showing the temperature dependence of pK values for the 18 buffers is presented. Proteins 33:159–166, 1998. © 1998 Wiley-Liss, Inc.     

The nature of spontaneous changes in growth rate in isolated coleoptile segments

About 4 hours after they are cut from the seedling, corn (Zea mays L.) coleoptile segments mounted vertically show a strong increase in growth rate. This increase occurs in water or various buffers near pH 7 and is not accompanied by the accumulation of a growth promoter in the medium. The increase in growth rate is prevented by 1 mmp-fluorophenylalanine and is strongly inhibited by 0.1 mmp-chlorophenoxyisobutyric acid.The increased growth rate is accompanied by a 95% increase in the ability of tissue extracts to catalyze the conversion of (14)C-tryptophan to (14)C-indole-3-acetic acid and by a nearly 3-fold increase in indole-3-acetic acid oxidase activity. The increase in growth rate is also observed in segments from coleoptiles grown aseptically.The spontaneous increase in growth rate is completely but reversibly inhibited by 1 mum indole-3-acetic acid. Cytokinins have little effect on the spontaneous growth response, whereas gibberellic acid is observed to extend the latent period and reduce the magnitude of the response. It is tentatively concluded that the increase in endogenous growth rate may result from increased auxin production upon derepression of the auxin biosynthesis pathway after isolating the tissue from the normal supply of auxin from the tip.     

铁矿开采对褐马鸡种群的影响

在山西五鹿山自然保护区的核心区就铁矿开采对褐马鸡(Casoptilon mantchuricum)种群的影响进行了调查。将铁矿周围的栖息环境分为<50 m、50-100 m和>100 m 3个分布带。开采前期,3个分布带中都可以遇见褐马鸡;开采初期,在<50 m分布带中已经看不到褐马鸡的踪迹,50-100 m分布带中的数量也有所减少,而在>100 m的分布带中褐马鸡的数量却有所增加,这表明由于铁矿开采的影响,褐马鸡从距铁矿较近的<50 m和50-100 m的分布带中,被迫转移到>100 m的分布带中活动。在铁矿开采3年以后,<50 m和50-100 m分布带中的遇见数都降至0,而>100m分布带中遇见的个体与开采初期相比,也有所下降。在开采初期,由于铁矿的数量较少,对褐马鸡种群活动综合影响并不显著,其种群密度并未发生明显变化。随着铁矿数量的增多和开采时间的延长,原来许多的觅食地,如林缘灌丛和地表草本都遭到破坏,而且铁矿开采的嘈杂声和爆破声此起彼伏,严重影响了褐马鸡正常的觅食活动。与开采初期相比,这种限制作用愈益显著,褐马鸡种群密度和空间分布也发生较大变化。研究结果表明,褐马鸡已不能适应铁矿开采带来的不良影响,在一些铁矿开采时间较长和数量较多的局部区域,褐马鸡已无法生存。     

Inhibition of protein carbamylation in urea solution using ammonium-containing buffers

Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times, and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium-containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium-containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium-containing buffers was developed to facilitate its application in proteomic research.     

The K+-H+ exchanger, nigericin, modulates taste cell pH and chorda tympani taste nerve responses to acidic stimuli

The relationship between acidic pH, taste cell pH(i), and chorda tympani (CT) nerve responses was investigated before and after incorporating the K(+)-H(+) exchanger, nigericin, in the apical membrane of taste cells. CT responses were recorded in anesthetized rats in vivo, and changes in pH(i) were monitored in polarized fungiform taste cells in vitro. Under control conditions, stimulating the tongue with 0.15 M potassium phosphate (KP) or 0.15 M sodium phosphate (NaP) buffers of pHs between 8.0 and 4.6, KP or NaP buffers did not elicit a CT response. Post-nigericin (500 × 10(-6) M), KP buffers, but not NaP buffers, induced CT responses at pHs ≤ 6.6. The effect of nigericin was reversed by the topical lingual application of carbonyl cyanide 3-chloro-phenylhydrazone, a protonophore. Post-nigericin (150 × 10(-6) M), KP buffers induced a greater decrease in taste cell pH(i) relative to NaP buffers and to NaP and KP buffers under control conditions. A decrease in pH(i) to about 6.9 induced by KP buffers was sufficient to elicit a CT response. The results suggest that facilitating apical H(+) entry via nigericin decreases taste cell pH(i) and demonstrates directly a strong correlation between pH(i) and the magnitude of the CT response.     

Characterization of polymeric buffers for operating membrane-trapped enzyme reactors in an electric field

A novel class of amphoteric, polymeric buffers, is described, consisting of grafting onto growing polyacrylamide chains weakly acidic and basic acrylamido-monomers (called Immobilines; protolytic groups as N-substituents on the nitrogen of the amido bond), for operating a membrane-immobilized enzyme reactor (MIER) in an electric field. With these soluble, polymeric buffers, it is possible to operate the membrane reactor at any optimum of pH activity, for any given enzyme, in the pH 3-10 scale. Such buffers, being amphoteric, are confined in the enzyme reaction chamber by the same isoelectric trapping mechanism. The best buffers were found to be those polymerized in presence of 9% neutral monomer (acrylamide) and containing 20 mM Immobiline as buffering ion. To decrease their viscosity in solution, the polymeric buffers are synthesized at high temperatures (70 degrees C) and in presence of a chain-transfer agent. The weight average molecular size in these conditions has been found to be ca. 200,000 Da. These buffers exhibited excellent performance in a variety of enzyme reactions in the MIER, such as in the case of penicillin G acylase and histidine decarboxylase and were found to greatly stabilize enzyme activity, permitting operation of the MIER over extended periods of time. As an example, in a penicillin G acylase reactor, >75% enzyme activity was maintained over a 10-d cycle of operation, while with conventional buffers more than 90% inactivation was experienced over the same period of time. This novel class of macromolecular, amphoteric buffers could also be exploited in other types of conventional bioreactors not based on an isoelectric trapping mechanism.