31P nuclear magnetic resonance evidence for the regulation of intracellular pH by Ehrlich ascites tumor cells

The phenomenon of intracellular pH (pHin) regulation in cultured Ehrlich ascites cells was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. Measurements were made with a Bruker WH 360 wide bore NMR spectrometer at a 31P frequency of 145.78 MHz. Samples at a density of 10(8) cells ml-1 were suspended in a final volume of 2 ml of growth medium in 10 mm diameter NMR tubes. Intracellular pH was calculated from the chemical shifts of either intracellular inorganic phosphate (Piin) or intracellular 2- deoxyglucose-6-phosphate (2dG6Pin). The sugar phosphate was used as a pH probe to supplement the Piin measurements, which could not always be observed. When available, the pHin calculated from the Piin peak was identical within experimental error to the pHin calculated from the 2dG6Pin peak. Intracellular pH was measured to be more alkaline than the medium at an external pH (pHex) below 7.1. Typical values were pHin = 7.00 for pHex = 6.50. These measurements were constant for times up to 165 min using well-energized, respiring cells. This pH gradient was seen to collapse immediately upon onset of anaerobic shock. Above a pHex of 7.2 there was no significant difference between pHin and pHex. These results unequivocally demonstrate the steady state nature of the pH regulation and its dependence upon energization.     

Compaction and particle segregation in myelin membrane arrays

Compacted membrane arrays are formed in the nerve myelin sheath by lowering the water activity (through evaporation or immersion in hypertonic solutions of nonelectrolytes or monovalent salts) or by binding specific cations (Ca(++), La(+++), and tetracaine at concentrations above 5-10 mM). X-ray diffraction observations on intact, hydrated nerves treated to induce compaction provide a control to assess the significance of structural changes seen by freeze-fracture electron microscopy. Compaction inevitably leads to lateral segregation of particles away from the closely packed membrane arrays into contiguous normal, or slightly expanded, period arrays. In the particle-enriched layers, the E fracture face is more particle-dense than the P face, whereas no particles are found on either face in the compacted layers. Morphologically, compaction induced by the all-or-nothing, relatively irreversible action of specific cations cannot be distinguished from compaction to the same extent induced by the graded, reversible effects of nonelectrolytes. Compaction by sodium chloride resembles that by specific- cation binding in that the repeat period is independent of reagent concentration; but, like dehydration by nonelectrolytes, the extent of compaction is reversibly related to reagent concentration. Sodium chloride-compacted myelin can be distinguished morphologically by a lack of the elongated border particles at the boundary between smooth and particle-enriched membrane observed for other compacting treatments. Fracture faces in compacted arrays are not always smooth, but the unusual appearances can be duplicated in purified myelin lipid multilayers subjected to similar treatments, which indicates that the particle-free membrane fracture faces are uninterrupted lipid hydrocarbon layers. Correlation of x-ray diffraction and electron microscopy observations provides a direct basis for identifying the intramembrane particles with transmembrane protein. The transmembrane protein appears to play a significant role in maintaining the normal membrane separation; swelling of the particle-enriched arrays in myelin compacted by tetracaine at low ionic strength provides information about the charge distribution on the transmembrane protein. Swelling of the compacted arrays following irreversible particle segregation shows that the interaction properties of the particle-free membranes are similar to those of pure lipid multilayers. Compaction and the consequent particle segregation in lyelin results from conditions stabilizing close apposition of the lipid bilayers. Particle segregation in areas of close contact between other cell membranes may also be driven by interbilayer attractive forces.     

Monocaryotization of Cultures of Lenzites trabea (Pers.) Fr. and other Wood-destroying Basidiomycetes

Cultures of Lenzites trabea and some other basidiomycetes grownon nutrient agar containing high concentrations of sodium arsenateconsistently reverted from the dicaryotic to the monocaryoticcondition. This effect could also be induced by a wide varietyof other toxicants, including copper sulphate, zinc sulphate,sodium dichromate, sodium pentachlorophenate, creosote, crystalviolet, boric acid, and sodium taurocholate, but sodium arsenatewas the most reliable monocaryotizing agent, being effectivewith eight of the fourteen basidio-mycete species tested withit. The neohaplonts formed by sodium arsenate and other toxicantsmate normally with each other and with compatible monosporeisolates. The value of chemical monocaryotization as a techniquein fungal genetics and experimental taxonomy is discussed. Byusing the mating factor alleles as genetic markers, it was demonstratedthat when L. trabea is monocaryo-tized by sodium arsenate orcopper sulphate, only one of the two nuclei in the dicaryoncan be recovered in the neohaplonts, the direction of this completenuclear selection depending on the toxicant and the dicaryonconcerned. It was shown to occur in treated wood blocks duringroutine soil-block decay tests of inorganic wood preservativesand it is probably a frequent, though hitherto unrecognized,occurrence in the laboratory testing of wood preservatives andother fungicides against basidiomycetes.     

Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains

Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S. cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction. Copyright 1998 John Wiley & Sons, Inc.     

A Comparison of the Use of In Vitro-Transcribed and Native rRNA for the Quantification of Microorganisms in the Environment

Abstract Nearly full-length, small subunit (SSU) rRNA was transcribed in vitro from clones of SSU rDNA genes. Comparing the use of in vitro-transcribed and native rRNA indicated that, when in vitro-transcribed rRNA was used as a standard for quantitative hybridizations with oligonucleotide probes, the population was consistently underestimated. The population abundance was expressed as a percentage of specific target SSU rRNA (determined with a specific oligonucleotide probe), relative to the total SSU rRNA (measured with a universal probe). Differences in hybridization signals could be related to specific probe target locations and rRNA denaturation conditions, suggesting that higher order structure is important in quantitative membrane hybridizations. Therefore, in vitro-transcribed rRNA cannot always be used for the absolute quantification of microbial populations, but can be employed as a standard to quantify shifts in population abundance over time, and to compare community structure in various environments.     

The self-incompatibility phenotype in brassica is altered by the transformation of a mutant S locus receptor kinase

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.     

Fertility table and rate of population growth of the predator Supputius cincticeps (Heteroptera: Pentatomidae) on one plant of Eucalyptus cloeziana in the field

The reproductive potential and population growth (r_m and λ) of the predator Supputius cincticeps (Heteroptera: Pentatomidae) were evaluated using a life and fertility table. S. cincticeps was reared on one plant of Eucalyptus cloeziana in the field and fed with Tenebrio molitor (Coleoptera: Tenebrionidae) pupae. Females of this predator had a net reproductive rate (R₀) of 21.02 females/female; an intrinsic rate of population increase (r_m) of 0.041 and finite rate of increase (λ) of 1.042. This resulted in population growth of S. cincticeps in the eucalyptus plant with a doubling time of 17.01 days. This natural enemy can be reared under field conditions with alternative prey for use in biological control. Such individuals of S. cinticeps will be better adapted to field conditions when they are liberated.