K<Superscript>+</Superscript> transport in the caterpillar intestine epithelium: role of osmolytes for the K<Superscript>+</Superscript>-secretory capacity of the tobacco hornworm midgut

The midgut of the tobacco hornworm, Manduca sexta, actively secretes potassium ions. This can be measured as short-circuit current (I_sc) with the midgut mounted in an Ussing chamber and superfused with a high-K⁺ saline containing as its major osmolyte 166 mM sucrose. Iso-osmotic substitution of sucrose by non-metabolisable compounds (mannitol, urea, NaCl and the polyethylene glycols 200, 400 and 600) led to a dramatic, though reversible, drop in the current. Acarbose, a specific inhibitor of invertase (sucrase) in vertebrates and insects, had no detectable influence on I_sc. Unexpectedly, after replacing sucrose iso-osmotically with the saccharides glucose, fructose, trehalose or raffinose, the K⁺ current could no longer be supported. However, all osmolytes smaller than sucrose (except for NaCl), metabolisable or not, initiated an immediate, quite uniform but transient, increase in I_sc by about 20%, before its eventual decline far below the control value. Hypo-osmotic treatment by omission of sucrose also transiently increased the K⁺ current. Small osmolytes substituted for sucrose caused no transient I_sc stimulation when the epithelium had been challenged before with hypo-osmolarity; however, the eventual decline in I_sc could not be prevented. Our data seem inconsistent with a role of sucrose as energiser or simple osmolyte. Rather, we discuss here its possible role as analogous to that of sucrose in lower eukaryotes or plants, as an extra- and/or intracellular compatible osmolyte that stabilises structure and/or function of the proteins implicated in K⁺ transport.Communicated by G. Heldmaier     

FTIR study of state and phase transitions of low moisture sucrose and lactose

Mid-infrared spectra of freeze-dried sucrose and lactose systems were acquired over a range of temperatures (30-200 degrees C) and water contents (0-6.3%). Starting from the glassy state, the experimental conditions were selected to cover the main thermal transitions: the glass-rubber transition, the crystallisation and, for some samples, the subsequent melting. The FTIR spectra were very sensitive to the physical state. While subtle but systematic spectral differences between the glassy and rubbery states were detectable throughout the spectrum, a very pronounced increase in spectral resolution was observed as crystallisation occurred and was followed by the expected spectral broadening during melting. The temperatures at which these changes occurred were in satisfactory agreement with the transition temperatures measured by differential scanning calorimetry (DSC). The increase in molecular mobility as a result of increasing temperature or plasticisation by water led to a significant shift of the O-H stretching band to higher wavenumbers indicating a weakening of hydrogen bonding. This shift reached a maximum as the DSC measured crystallisation temperature range was approached. As expected, the crystallisation led to a highly effective hydrogen bonding network. This was more significant for lactose than for sucrose. No significant step change in hydrogen bonding was observed at Tg. As anticipated, the temperature at which these transitions occurred decreased with increasing water content but overlapped when observed in the context of the shifted temperature (T-Tg).     

Effect of iron concentration on hydrogen fermentation

The effect of the iron concentration in the external environment on hydrogen production was studied using sucrose solution and the mixed microorganisms from a soybean-meal silo. The iron concentration ranged from 0 to 4000 mgFeCl₂ l⁻¹. The temperature was maintained at 37°C. The maximum specific hydrogen production rate was found to be 24.0 mlg⁻¹ VSSh⁻¹ at 4000 mgFeCl₂ l⁻¹. The specific production rate of butyrate increased with increasing iron concentration from 0 to 20 mgFeCl₂ l⁻¹, and decreased with increasing iron concentration from 20 to 4000 mgFeCl₂ l⁻¹. The maximum specific production rates of ethanol (682 mgg⁻¹ VSSh⁻¹) and butanol (47.0 mgg⁻¹ VSSh⁻¹) were obtained at iron concentrations of 5 and 3 mgFeCl₂ l⁻¹, respectively. The maximum hydrogen production yield of 131.9 mlg⁻¹ sucrose was obtained at the iron concentration of 800 mgFeCl₂ l⁻¹. The maximum yields of acetate (389.3 mgg⁻¹ sucrose), propionate (37.8 mgg⁻¹ sucrose), and butyrate (196.5 mg g⁻¹ sucros) were obtained at iron concentrations of 3, 200 and 200 mgFeCl₂ l⁻¹, respectively. The sucrose degradation efficiencies were close to 1.0 when iron concentrations were between 200 and 800 mgFeCl₂ l⁻¹. The maximum biomass production yield was 0.283 gVSSg⁻¹ sucrose at an iron concentration of 3000 mgFeCl₂ l⁻¹.     

Production of poly-β-hydroxybutyrate by thermophilic cyanobacterium, Synechococcus sp. MA19, under phosphate-limited conditions

Synechococcus sp. MA19, grown autotrophically under phosphate-limited conditions at 50 °C, produced poly--hydroxybutyrate (PHB) when intracellular phosphate content was 0.043–0.076mmol per g of cellular components. In the culture for 260h using Ca₃(PO₄)₂ as a phosphate source, strain MA19 accumulated PHB at 55% (w/w) of the dry cells and the amount of PHB produced was 2.4gl^–1 which was almost twice that without Ca₃(PO₄)₂ addition.     

Effect of the respiratory activity on photoinhibition of the cyanobacterium Synechocystis sp.

The influence of respiratory activity on photosynthesis in Synechocystis cells that had been exposed to high light intensity was studied using distinct conditions of nitrogen supply. The photoinhibitory rate of N-sufficient cells was not influenced by the presence of different nitrogen sources. In contrast, when N-starved cells were resupplied with ammonium, they were protected from photoinhibition. Although N-starved cells presented a higher rate of dark O₂ uptake than N-sufficient ones, the photoinhibitory rate increased in both cases after addition of sodium azide or sodium azide plus salicylhydroxamic acid in the photoinhibitory treatment. In the absence of the D₁ protein repair mechanism, photodamage to Photosystem II was faster in N-sufficient cells than in N-starved ones. Mitigation of photodamage disappeared when the respiratory activity of N-starved cells was partially suppressed by the addition of sodium azide or sodium azide and salicylhydroxamic acid. Our results suggest that electron flow through cyanobacterial terminal oxidases can assist Photosystem I in removing electrons from the reduced plastoquinone pool, thus contributing to both reopening of Photosystem II reaction centers and avoiding photogeneration of reactive oxygen species under photoinhibitory conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

A technique for isolation of rubella virus-like particles by sucrose gradient ultracentrifugation using Coomassie brilliant blue G crystals

An improved method for the isolation of rubella virus-like particles (RVLP) from cell culture supernatant of transfected Chinese hamster ovary (CHO24S) cells is described. It employs a combination of membrane filtration with sucrose gradient ultracentrifugation. It was found that staining the RVLP band with Coomassie brilliant blue G (CBB) resulted in the CBB crystals adsorbing RVLP. After ultracentrifugation (25,000 rpm, 3h, 4 degrees C) a sharp blue band with crystals (diameter 30-40 microm) was observed (at a density of 1.250 g/ml at 25 degrees C) in a 30-60% sucrose gradient. Using a combination of SDS-PAGE and Western blotting techniques, E1 rubella virus structural protein was detected only in the solutions derived from the sharp blue band. A decrease in crystal concentration a few millimeters above or below the main band was associated with a decrease in protein concentration. By dilution with a saturated ice-cold 30% sucrose solution it was possible to pellet the crystals by centrifugation (15,000 rpm, 10 min). SDS-PAGE showed a much higher concentration of RVLP structural protein in the pellet than in the supernatant. This RVLP-containing material is especially suitable for the preparation of rubella virus immunoblot stripes.     

Factors influencing the growth of micropropagated shoots and in vitro flowering of gentian

About 70% of the shoots developed from nodal explants ofGentiana triflora flowered in vitroondouble strength WPM medium containing 3% (w/v) sucrose, 0.5mg/l BA after 12 weeks of culture in a growth room at 22°Cwith continuous illumination (PPFD=60molm^–2 s^–1). The influences oninvitro shoot development and flowering of several factors includingthe position of the explant, requirements for sucrose, cytokinin orGA₃, variations of pH and photosynthetic photon flux density (PPFD)were investigated. In vitro flowering but not shootdevelopment of G. triflora decreased notably withincreaseddistance from the apex of the shoot, indicating the presence of a floralgradient in the micropropagated shoots. Conversely, as little as 0.01mg l^–1 GA₃ in the medium promotedshootdevelopment but even up to 0.2 mg l^–1GA₃ did not induce in vitro flowering.Even though BA could substitute GA₃ for a high level of shootdevelopment, it also promoted a high level of in vitroflowering at the PPFD of 60 molm^–2 s^–1. Sucrose was required for shootdevelopment and flowering in vitro and higher levels ofPPFD could not compensate effectively for the omission of the sugar from themedium. In general, the effects of different concentrations of BA in the mediumor variations of pH on shoot development and flowering invitro were found to be influenced by PPFD. A novel observation isthat precocious flowering of micropropagated gentian shoots did not occur ifthey were first cultured for 5 weeks in the dark before transfer to the lightcondition.     

Phytohormone-mediated transformation of sugars to starch in relation to the activities of amylases, sucrose-metabolising enzymes in sorghum grain

Indole-acetic acid (IAA) and abscisic acid (ABA) were fed throughcomplete liquid medium (containing 2, 4, 8% sucrose) to detached earheads of sorghum. The effect of these phytohormones on interconvertion ofsugarsand their transformation to starch in relation to the activities of-, -amylases, sucrose-synthase (synthesis), sucrose-phosphatesynthase and soluble invertases was studied in the grain. This effect on theuptake of (U-¹⁴C) sucrose by detached ear heads and incorporation of¹⁴C into free sugars and starch of grain and into free sugars ofinflorescence parts was also studied. At concentrations of up to 4%sucrose in the culture medium, IAA increased the content of total free sugarsinthe grain. However, accumulation of starch and activities of - and-amylases increased when lAA was present even beyond the 4%sucroseconcentration in the culture medium. At all sucrose concentrations, the effectsof ABA and IAA were opposite. With 4% sucrose, both phytohormones causedmaximum accumulation of starch in the grain. ABA enhanced the relativeproportion of sucrose in the sugar pool with a concomitant reduction in theactivities of soluble acid (pH 4.8) and neutral (pH 7.5) invertases. Incontrast, IAA decreased the sucrose proportion of grain sugars with asimultaneous elevation and reduction in the activities of invertases andsucrose-phosphate synthase, respectively. Irrespective of sucrose concentrationin the culture medium, the activity of sucrose synthase (synthesis) wasenhancedwith IAA as well as ABA at their 10 M concentration. IAA alsoenhanced incorporation of ¹⁴C from (U-¹⁴C) sucrose intothe EtOH extract (principally constituted by free sugars) and starch of thegrain, but ABA caused the reverse effect. Based on the results, it is suggestedthat IAA and ABA have contrasting effects on the transformation of sucrose tostarch in sorghum grain where its capacity to synthesise starch is modulatedpositively by IAA and negatively by ABA.