Production of parvovirus B19 vaccine in insect cells co-infected with double baculoviruses

Recombinant human parvovirus B19 virus-like particles (VLPs), a candidate vaccine, were produced using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. The synthesis and assembly of the particles in Sf-9 cells are directed by double infections with one recombinant virus (bacVP1) expressing the parvovirus minor viral protein VP1 and a second virus (bacVP2) expressing the major viral protein VP2. Previous animal studies demonstrated that the polypeptide composition of the VLPs strongly affects the elicitation of virus neutralizing antibodies. The key factor controlling the production of an immunologically potent product in bioreactors was identified to be the multiplicity of infection (MOI) of bacVP1 and bacVP2 used for infection. A probabilistic model, which correlates well with the experimental results, was employed to facilitate the selection of MOIs and to provide a better understanding of the baculovirus co-infection process. A novel production process based on secondary infections was developed to ensure product consistency and to simplify large-scale logistics. The effects of other critical process parameters, such as temperature, dissolved oxygen concentration, lactate concentration, cell concentration at infection, and harvest time, were also investigated. (c) 1996 John Wiley & Sons, Inc.     

Four-million-year-Old hominids from East Lake Turkana,Kenya

A piece of mandible and several isolated teeth are reported from fluviatile sediments older than 4 million years at East Lake Turkana. They most closely resemble hominids from Laetoli, Tanzania and Hadar, Ethiopia which have been assigned to Australopithecus afarensis. © 1994 Wiley-Liss, Inc.     

Genetic differentiation between Carex lasiocarpa and C. Pellita (Cyperaceae) in north america

Carex lasiocarpa and C. pellita (sect. Carex) share a very similar morphology and have overlapping ranges in North America, but are found in different habitats characterized by contrasting soil types and pH. We studied allozyme variation and chromosome numbers to assess genetic differentiation between the two taxa. Both principal components analysis on the allele frequencies from 12 putative enzyme-coding loci and cluster analysis of genetic identities separated 51 sampled populations into two groups that were consistent with recognized structural differences between C. lasiocarpa and C. pellita. Mean within-group genetic identities were 0.95 for C. lasiocarpa and 0.93 for C. pellita; mean between-group genetic identity was 0.81. With the exception of two rare alleles, the alleles of C. pellita were a subset of those found in C. lasiocarpa. Principal components analysis of measurements of structural characters from voucher specimens representing 46 populations also separated the two species with minimal overlap. Meiotic squashes of microsporocytes revealed haploid chromosome numbers of 38 and 38 + 1 for C. lasiocarpa and 41 and 40 + 1 for C. pellita. These data support the continued recognition of the two taxa as distinct species, and suggest that C. pellita may be a daughter species still in the process of divergence from C. lasiocarpa.     

Costs of reproduction in the pink lady's slipper orchid (Cypripedium acaule): defoliation,increased fruit production,and fire

An earlier experiment with the pink lady's slipper orchid demonstrated that plant leaf area was lowered only after successive years of increased fruit production. This result suggested that the cost of reproduction was small in relation to the energy budget of the plant. To test this idea, plants were subjected to experimental hand-pollination treatments to increase fruit set as well as leaf removal treatments to decrease the energy budget of plants. Changes in plant size in years 2 and 3 and, to some extent, rate of flowering, were determined by a combination of initial plant size, leaf removal treatments in year 1, fruit production in year 1, and damage from an unplanned fire in year 2. Plants that had both leaves removed and produced a fruit in 1987 decreased in size in the following 2 years in comparison with other treatment groups. The cost of fruit production was not apparent in plants that had only one or no leaves removed. Plants apparently have to be put into severe physiological stress in order for a cost of reproduction to appear in the following year. The cost of producing one fruit was a decline of plant size in the following year of 30 cm², which is very similar to our previous experiment using a different design. An additional experiment failed to find evidence that these plants increase their photosynthetic rate to compensate for the loss of leaves or the cost of maturing fruit. Published experiments in both the greenhouse and the field that failed to find a cost of reproduction should be reevaluated in terms of the intensity of treatment imposed and the overall energy budget of the plant in field situations.     

Activation of TNF-R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-α has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling. © 1994 Wiley-Liss, Inc.     

Soil pCO2, soil respiration,and root activity in CO2-fumigated and nitrogen-fertilized ponderosa pine

The purpose of this paper is to describe the effects of CO₂ and N treatments on soil pCO₂, calculated CO₂ efflux, root biomass and soil carbon in open-top chambers planted with Pinus ponderosa seedlings. Based upon the literature, it was hypothesized that both elevated CO₂ and N would cause increased root biomass which would in turn cause increases in both total soil CO₂ efflux and microbial respiration. This hypothesis was only supported in part: both CO₂ and N treatments caused significant increases in root biomass, soil pCO₂, and calculated CO₂ efflux, but there were no differences in soil microbial respiration measured in the laboratory. Both correlative and quantitative comparisons of CO₂ efflux rates indicated that microbial respiration contributes little to total soil CO₂ efflux in the field. Measurements of soil pCO₂ and calculated CO₂ efflux provided inexpensive, non-invasive, and relatively sensitive indices of belowground response to CO₂ and N treatments.     

Influence of pruning management on P and N distribution and use efficiency by N2 fixing and non-N2 fixing trees used in alley cropping systems

Pruning of hedgerow trees is an important management practice for the successful establishment of an alley cropping system. Although pruning affects biomass production, only meager evidence of this management on distribution of nutrients among the different plant organs after tree regrowth is available. This study examined the effect of pruning on the distribution and use efficiency of N and P in a N₂ fixing leguminous tree species, Gliricidia sepium, and two non-N₂ fixing leguminous tree species, Senna siamea and S. spectabilis, grown in a field on an Alfisol (low in P) at Fashola (Guinea Savanna Zone), Southwestern Nigeria. Four P rates, 0, 20, 40 and 80 kg P ha^–1 as single superphosphate were used and management treatments included pruned versus unpruned plants. The ¹⁵N isotope dilution technique was used to measure N₂ fixation in G. sepium. Partitioning of total P among different plant organs was influenced by plant species and pruning management, but was not affected by P application rates. The distribution of total P in the various plant organs followed that of dry matter yield while N partitioning had a different pattern. Pruned plants distributed about 118% more total P to branches and had a higher physiological P use efficiency (PPUE) than unpruned plants. Leaves were the biggest sink for total N and N allocation in the other plant organs was influenced by plant species and pruning management, G. sepium had relatively more of its total N and P partitioned into roots (about double that of the non-N₂ fixing trees) but had a lower PPUE. Unpruned and pruned G. sepium derived 35 and 54% respectively of their total N from atmospheric N2, with about 54% of the fixed N2 being allocated to leaves and roots. Results showed that N and P pools turned over in the branches during plant regrowth after pruning but the causative factors associated with this phenomenon were not clear.     

Protein Synthesis and Breakdown during Heat Shock of Cultured Pear (Pyrus communis L.) Cells

Cultured pear (Pyrus communis L. cv Passe Crassane) cells were subjected to temperatures of 39, 42, and 45[deg]C. Heat-shock protein (hsp) synthesis was greater at 30[deg]C than at temperatures above 40[deg]C and continued for up to 8 h. Both cellular uptake of radiolabeled methionine and total protein synthesis were progressively lower as the temperature was increased. Polysome levels decreased immediately when cells were placed at 39 or 42[deg]C, although at 39[deg]C the levels began to recover after 1 h. In cells from both temperatures, reassembly occurred after transfer of cells to 25[deg]C Four heat-shock-related mRNAs[mdash]hsp17, hsp70, and those of two ubiquitin genes[mdash]all showed greatest abundance at 39[deg]C and decreased at higher temperatures. Protein degradation increased with time at 42 and 45[deg]C, but at 39[deg]C it increased for the first 2 h and then decreased. In the presence of cycloheximide, which prevented hsp synthesis, protein degradation at 39[deg]C was as great as that at 45[deg]C in the absence of cycloheximide. The data suggest that hsps may have a role in protecting proteins from degradation at the permissive temperature of 39[deg]C. At temperatures high enough to inhibit hsp synthesis, protein degradation was enhanced. Although ubiquitin may play a role in specific protein degradation, it does not appear to be involved in increased protein degradation occurring above 40[deg]C.     

Distinct morphological and mito-inhibitory effects induced by TGF-β1, HGF and EGF on mouse,rat and human hepatocytes

TGF-1 is known as a potent inhibitor of proliferation of rat and human hepatocytes. In this study we show that the effects of TGF-1 are quite different on mouse hepatocytes. In rat and human hepatocytes, TGF-1 inhibited DNA synthesis and also inhibited the morphological changes induced by growth factors in rat and human hepatocytes. In contrast, addition of TGF-1 to mouse hepatocytes resulted in pronounced alterations in morphology of these cells. These changes were similar to those induced by HGF and EGF. The induction of structural changes by TGF-1 was noted only in mouse hepatocytes. Mouse hepatocytes were also much more resistant to the mito-inhibitory effect of TGF-1. These findings suggest profound differences in hepatocyte growth regulation between these species and may relate to observed differences in susceptibility to carcinogenesis.Abbreviations EGF epidermal growth factor - HGF hepatocyte growth factor - SF scatter factor - TGF-1 transforming growth factor beta type one     

Fine root growth and demographic responses to nutrient patches in four old-field plant species

Proliferation of roots in a nutrient patch can occur either as a result of an increase in root length (morphological response) or by a change in root birth or death rates (demographic responses). In this study we attempted to distinguish between these two mechanisms of response to nutrient patches and to compare the responses of four old-field plant species (two annuals, two perennials). For all four species combined, there were significant increases in root numbers and root length in fertilized patches. Root proliferation in fertilized patches was largely due to increased birth (=branching) rates of new roots. However, there was also a significant increase in root death rates in the fertilized patches which reduced the magnitude of the increase in net root numbers. Plots for individual species suggested they differed in the magnitude and timing of root proliferation in fertilized patches due to differences in root birth and death rates. However, because of the limited sample size in this study, there was only a marginally significant difference among species in root birth rates, and no difference in death rates. Further studies are currently underway to better quantify species differences in the demographic mechanism, as well as magnitude, of response to nutrient patches and if this would affect the ability to exploit small-scale heterogeneity in soil resources.