Genetic engineering to contain the Vitreoscilla hemoglobin gene enhances degradation of benzoic acid by Xanthomonas maltophilia

Xanthomonas maltophilia was transformed with the gene encoding Vitreoscilla (bacterial) hemoglobin, vgb, and the growth of the engineered strain was compared with that of the untransformed strain using benzoic acid as the sole carbon source. In general, growth of the engineered strain was greater than that of the untransformed strain; this was true for experiments using both overnight cultures and log phase cells as inocula, but particularly for the latter. In both cases the engineered strain was also more efficient than the untransformed strain in converting benzoic acid into biomass. (c) 1996 John Wiley & Sons, Inc.     

META-ANALYSES OF THE ASSOCIATION BETWEEN MULTILOCUS HETEROZYGOSITY AND FITNESS

Meta-analyses of published correlation coefficients between multilocus heterozygosity (MLH) and two fitness surrogates, growth rate and fluctuating asymmetry, suggested that the strength of these correlations are generally weak. A variety of plants and animals was included in the meta-analyses. A statistically homogeneous group of MLH–growth rate correlation coefficients that included both plants and animals yielded a common correlation of r_z = 0.133. A common correlation of r_z = –0.170 was estimated for correlations between MLH and fluctuating asymmetry in three species of salmonid fishes. These results suggest that selection, including overdominance, has at most a weak effect at allozyme loci and cast some doubt on the widely held notion that heterozygosity and individual fitness are strongly correlated.     

Hydrostatic Pressure Has Different Effects on the Assembly of Tubulin, Actin, Myosin II, Vinculin, Talin, Vimentin, and Cytokeratin in Mammalian Tissue Cells

Hydrostatic pressures in the range of hundreds of atmospheres are known to disrupt cytoskeletal organization in tissue culture cells, with profound changes in cell shape. The molecular mechanisms of these effects are poorly understood. To determine the effect of pressure on the cytoskeleton, and thus to provide better indicators of the molecular mechanisms, we used fluorescent antibody staining to compare the organizations of seven different cytoskeletal proteins in HeLa cells and rat osteosarcoma cells (ROS-17/2.8) subjected to different pressures up to 400 atm. Pressures of 300 atm or more caused cells of both lines to “round up” and to withdraw their lamellar extensions. However, this response varied within a population of cells, with some cells remaining spread at pressures that caused their neighbors to round up. The most resistant to rounding were those cells touching other cells, and the occasional giant cells. As expected, the rounded cells showed disruption of actin stress fibers and of vinculin and talin at focal contacts. The unrounded cells showed less disruption in the organization of these same proteins. Microtubules and myosin II filaments appeared resistant to 400 atm pressure in both cell types, whether rounded or unrounded. However, in HeLa cells, the intermediate filaments, vimentin and cytokeratin, depolymerized and formed small vesicles when pressures exceeded 200 atm, and this occurred in rounded as well as unrounded cells. In osteosarcoma cells, which do not have cytokeratin, vimentin did not depolymerize. We discuss different mechanisms that might explain these responses to pressure, including direct effects on the equilibria of protein polymerization and less direct effects on regulatory mechanisms, such as phosphorylation pathways, that control cytoskeletal organization. The latter type of explanation seems more consistent with both the variability of response within cell populations and the difference in vimentin's response in one cell line compared with the other.     

PHOTOSYNTHESIS AND REGULATION OF RUBISCO ACTIVITY IN NET PHYTOPLANKTON FROM DELAWARE BAY1

Net phytoplankton (> 20 μm) comprised 51 ± 9% of the total chlorophyll (Chl) in a Skeletonema costatum– dominated spring bloom in Delaware Bay. The net phytoplankton had low C:N and high protein: carbohydrate ratios, indicating that their growth was nutrient-replete. Their photosynthetic responses were characterized by low specific absorption, low light-limited and light-saturated rates of photosynthesis, and high quantum yields, indicative of acclimation to low irradiance and internal self-shading. High fucoxanthin: Chi ratios also indicated low light acclimation, but high photoprotective xanthophyll: Chi ratios suggested a high capacity for photoprotective energy dissipation. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) could be activated and deactivated in response to changes in irradiance and was fully activated at the surface of the water column and fully deactivated in aphotic deep water. Maximum Rubisco activity was correlated with Rubisco content and bulk protein content of the phytoplankton and with light-saturated rates of photosynthesis measured in short (< 20-min) incubations. Long (60-min) incubations caused a decrease in the light-saturated rate of photosynthesis, possibly because of feedback limitation. While feedback limitation is unlikely to occur in the water column, it should be considered when estimating productivity in well-mixed waters from fixed light-depth incubations.     

Light regulation of phosphoenolpyruvate carboxylase in barley mesophyll protoplasts is modulated by protein synthesis and calcium, and not necessarily correlated with phosphoenolpyruvate carboxylase kinase activity

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca²⁺, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C₃ leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C₃ protoplasts than in C₄ protoplasts or leaf tissue.Abbreviations CAM Crassulacean acid metabolism - Chl chlorophyll - CHM cycloheximide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - PEPCase PEP carboxylase     

Enhancement by bacterial hemoglobin of amylase production in recombinant E. coli occurs under conditions of low O2

Summary In shake flask growth, the presence of Vitreoscilla hemoglobin (VHb) enhanced production of recombinant -amylase in E. coli by about 80% (generally confirming our previous results). Extension of these studies to growth in oxygen controlled fermentors showed that VHb afforded no advantage in -amylase production when oxygen was not limiting, but resulted in about a 6-fold increase when oxygen was limiting. This increase was due almost entirely to secreted enzyme.     

MEASURING THE EFFECTS OF PAIRING SUCCESS,EXTRA-PAIR COPULATIONS AND MATE QUALITY ON THE OPPORTUNITY FOR SEXUAL SELECTION

Sexual selection can act through variation in the number of social mates obtained, variation in mate quality, or variation in success at obtaining extra-pair fertilizations. Because within-pair fertilizations (WPF) and extra-pair fertilizations (EPF) are alternate routes of reproduction, they are additive, rather than multiplicative, components of fitness. We present a method for partitioning total variance in reproductive success (a measure of the opportunity for selection) when fitness components are both additive and multiplicative and use it to partition the variance into components that correspond to each mechanism of sexual selection. Computer simulations show that extra-pair fertilizations can either increase or decrease total variance, depending on the covariance between within-pair and extra-pair success. Simulations also suggest that for socially monogamous species, extra-pair fertilizations have a greater effect than variation in mate quality or pairing status on the opportunity for selection. Application of our model to data gathered for a population of red-winged blackbirds (Agelaius phoeniceus) indicates that most of the variance in male reproductive success was attributable to within-pair sources of variance. Nevertheless, extra-pair copulations increased the opportunity for selection because males varied both in the proportion of their social young that they sired and in the number of extra-pair mates that they obtained. Furthermore, large and positive covariances existed between the number of extra-pair mates a male obtained and both social pairing success and within-pair paternity, indicating that, in this population, males preferred as social mates also were preferred as extra-pair mates.     

Impairment of multicycle influenza virus growth in Vero (WHO) cells by loss of trypsin activity.

We demonstrated that influenza virus replication in Vero (WHO) cells, a subline of African green monkey kidney cells, is impaired by rapid inactivation of trypsin in the culture fluids. Trypsin inactivation was caused by a factor secreted by Vero cells into the media. Repeated addition of trypsin to the culture medium of influenza virus-infected Vero cells restores the multicycle growth pattern of influenza A virus strains, allowing high yields to be obtained at a low multiplicity of infection. These findings may permit efficient use of Vero (WHO) cells in the production of influenza vaccines.     

Selection of a single amino acid substitution in the hemagglutinin molecule by chicken eggs can render influenza A virus (H3) candidate vaccine ineffective.

This study investigated whether a single amino acid change in the hemagglutinin (HA) molecule influenced the efficacy of formalin-inactivated influenza A (H3N1) vaccine candidates derived from high-growth reassortants between the standard donor of high-yield genes (A/PR/8/34 [H1N1]) and host cell variants generated from the same clinical isolate (A/Memphis/7/90 [H3N2]) by passage in embryonated chicken eggs. Two clones of the isolate generated by growth in eggs differed from the parent virus (represented by an MDCK cell-grown counterpart) solely by the presence of Lys (instead of Glu) at position 156 or Ile (instead of Ser) at position 186 in the HA1 subunit. The protective efficacy of egg-grown HA Lys-156 and HA Ile-186 reassortant variants was compared with that of the MDCK cell-grown reassortant vaccine. Classically, antibody titers in serum have been used to demonstrate vaccine efficacy. Here, parameters of B-cell responsiveness were monitored, including the kinetics, character, and localization of the primary antibody-forming cell (AFC) response and the development of B-cell memory in lymphoid tissues associated with the priming site (spleen) and responsive to pulmonary challenge with infectious virus (upper and lower respiratory tract lymph nodes). We show that the egg-grown HA Lys-156 variant induced an AFC profile vastly different from that elicited by the other two reassortant vaccines. The vaccine was poorly immunogenic; it induced antibodies that were cross-reactive prior to challenge but which, postchallenge with a lethal dose of the MDCK cell-grown reassortant virus, were targeted primarily to the HA Lys-156 variant, were of the immunoglobulin M isotype, were nonprotective, and were derived from the spleen. In contrast, the egg-grown HA Ile-186 variant was remarkably like the MDCK cell-grown virus in that protective immunoglobulin G antibodies were unaffected by the Ile-186 substitution but poorly recognized HA with Lys-156. Furthermore, memory AFC responsiveness was localized to regional lymphoid tissue in the upper respiratory tract, where challenge HA was found. Thus, it is recommended that in the selection of vaccine candidates, virus populations with the egg-adapted HA Lys-156 substitution be eliminated and that, instead, egg-grown isolates which minimally contain Ile-186 be used as logical alternatives to MDCK cell-grown viruses.     

The influence of stand development on nutrient demand,growth and allocation

As an even-aged stand develops growth is concentrated first on leaves and fine roots, as a result nutrient accumulation is very rapid. During this early stage there is a distinct species effect whereas later nutrient uptake becomes a function of growth rate irrespective of species. Once canopy is closed up to two thirds of the nutrients required for growth can be obtained by retranslocation from older or dying tissues, an efficient conservation mechanism that leads to a reduction in the demands that are further reduced by the cycle through the litter layer. In consequence nutritional problems are most likely in the early years while the green crown is being constructed. Later in the rotation problems are unlikely unless nutrient cycles are disturbed, for example by thinning or as a result of excessive accumulation of humus. The eventual clear felling is a major disruption to nutrient cycles. Accelerated litter decomposition can lead to leaching losses, although this can be short lived, and burning if practised can have a major impact on poor sites. Nutrient loss in material removed from the felling site, whether or not harvested, is not high but is much increased if crowns are removed, particularly for the heavily crowned species. The importance of such loss clearly varies with site but may be significant for more than just loss of nitrogen, with loss of calcium, phosphorus or even organic matter per se all being possibly causes of worry.