Alternating bolus and continuous infusion 5-fluorouracil: a strategy to overcome resistance to this fluoropyrimidine in advanced colorectal cancer patients

Focusing our effort on the importance of FUra scheduling we have tested the hypothesis that pulse and continuous infusion (CI) of the fluoropyrimidine have different mechanisms of cytotoxicity. Our initial approach was to compare the mechanism of resistance of a cell line resistant to a short term exposure to FUra (HCT-8/FU4hR) to that of a cell line resistant to a prolonged exposure to the fluoropyrimidine (HCT-8/FU7dR). Cytotoxicity studies showed that HCT-8/FU4hR cells were still sensitive to FUra given as a 7-d exposure, suggesting different mechanisms of resistance. Indeed, rapid recovery of TS activity after drug removal was evident in the HTC-8/FU7dR cell line while HCT-8/FU4hR cells were similar to the parental cell line with regard to both the degree of in situ TS inhibition by FUra and duration of inhibition after FUra removal. In contrast, labelling studies with [³H-6] FUra (4 h exposure, 100 M) showed that the incorporation of the fluoropyrimidine into RNA is significantly decreased in HCT-8/FU4hR cells as compared to parental HCT-8 cells.Given the lack of cross resistance between the two schedulesin vitro, a pilot trial was done on patients with colorectal cancer refractory to bolus FUra. On 15 patients failing after FUra+LV or FUra alone 1 PR, 3 MR, 3 SD and 8 P were observed, confirmng a certain degree of activity of CI FUra in patients clinically resistant to bolus FUra.Based on this rationale, a phase II trial of schedule-oriented biochemical modulation of FUra in advanced colorectal cancer patients was conducted, employing a hybrid regimen of 2 biweekly cycles of FUra bolus (600 mg/sqm), preceeded by (24 h interval) methotrexate, 200 mg/sqm (in order to maximize the RNA effect of the drug) alternating with FUra continuous infusion, 200 mg/sqm daily for 3 weeks, modulated by leucovorin, 20 mg/sqm weekly bolus (in order to maximize the DNA effect).Thirty-three consecutive patients (median ECOG PS 1) with advanced measurable colorectal cancer and no prior therapy for metastatic disease entered the study, from February 1992 to August 1993. Three complete and 13 partial responses were obtained among these 33 patients (RR=48%, 95% confidence limis, 31–66%). After a median follow-up time of 23 months, 16 patients are still alive. The median progression free survival and overall survival were 9.6 and 20.8 months, respectively. No toxic deaths or grade 4 toxicity occurred. The incidence of grade 3 toxicity per patient in any cycle was: mucositis 6%, diarrhea 3% and vomiting 3% for the bolus part and 21%, 3% and 6% respectively, for the continuous infusion part of the regimen. Hand-foot syndrome occurred in 27% of the patients treated with the continuous infusion regimen.In conclusion, this experimental and clinical project has generated a novel regimen of schedule oriented biochemical modulation that is twice as active and half as toxic compared to bolus FU+LV given with either the daily x 5 or the weekly schedule. This high clinical activity is very encouraging, especially considering that 1) consecutive patients were entered, 2) the responses were independently reviewed, 3) the progression free survival and survival were much longer than those actually reported for this disease, 4) the toxicity of the program, in particular the bolus regimen, was relatively low allowing further intensification.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Analysis of the multi-stem clump structure ofLitsea japonica Juss. growing in a coastal dwarf forest

The multi-stem clump structure of a coastal dwarf forest dominated byLitsea japonica Juss. was investigated in order to clarify the sprouting characteristics and self-maintenance of clumps by stem alternation. The size and age distribution of multi-stem clumps were analyzed using cumulative relative frequency curves.L. japonica had a large number of stems and an even height distribution or young age-biased distribution of stems within a clump. These results indicated the sequential flushing of sprouts at high frequency. Height distribution within a clump ofL. japonica was relatively even compared to other species. This clump structure suggested the stable self-maintenance of individuals in all ranges of size and age without disturbances. It originated specific sprouting characteristics as a response to the severe stress of salty wind.Ardisia sieboldii Miq. had few stems within a clump. Although the stem height distribution of large individuals tended to be even, most clumps had a large size-biased distribution of stem height which indicated simultaneous sprouting. From this structure, sprouts of this species were thought to be of less significance in the stable self-maintenance of individuals thanL. Japonica.     

Invertebrate phosphatidylinositol-specific phospholipases C and their role in cell signaling

Phosphatidylinositol-specific phospholipase C (PLC) is a family of enzymes that occupy a pivotal role in one of the largest classes of cellular signaling pathways known. Mammalian PLC enzymes have been divided into four major classes and a variety of subclasses based on their structural characteristics and immunological differences. There have been five invertebrate PLC-encoding genes cloned thus far and these fall within three of the four major classes used in categorizing mammalian PLC. Four of these invertebrate genes have been cloned fromDrosophila melanogaster and one is fromArtemia, a brine shrimp. Structural characteristics of the invertebrate enzymes include the presence of highly conserved Box X and Box Y domains found in major types of mammalian PLC as well as novel features. Two of the invertebrate PLC genes encode multiple splice-variant subtypes which is a newly emerging level of diversity observed in mammalian enzymes. Studies of the invertebrate PLCs have contributed to the identification of the physiological functions of individual isozymes. These identified roles include cellular processes such as phototransduction, olfaction, cell growth and differentiation.     

Contribution of Monsoon Asia to the carbon budget of the biosphere,past and future

The High Resolution Biosphere Model (HRBM), which has been developed by the group of the author, was used to investigate the carbon balance of the vegetation and the soil in the ecosystems of Monsoon Asia in comparison to the rest of the world. The HRBM is a global grid-based (0.5 degree resolution) model with a monthly time step. It includes modules for natural vegetation, land use, vegetation fires, vegetation composition. A historical carbon budget was calculated for the period 1860–1978 and, on a global scale, validated using atmospheric CO₂ data. Based on the per-country development of the population and their requirements, different reasonable scenarios were used to investigate the potential impacts of land use and deforestation in the period 1990–2050. The HRBM calculates considerable contributions of Monsoon Asia to the global CO₂ emissions due to land use changes in the past. Between 1860 and 1978, about 1/4 of the global releases from land use changes came from South Asian and Southeast Asian biota. The future contributions in the period 1990–2050 depend on the assumed development of the agricultural methods. If the intensity of agriculture and the agricultural productivity will stay the same as in the 1980s, there will be a strong need to increase agricultural areas, and thus deforestation will dominate. If there will be a change over to intensive methods of agricultural production, the presently used areas might be sufficient to provide resources to the growing population.     

Using XAD resins to study the effects of reduced organic micropollutant concentrations in River Rhine and Meuse water on phytoplankton growth

Laboratory incubation experiments were used to study the effect of reduced concentrations of organic micropollutants in water from the rivers Rhine and Meuse on the specific growth rate of the river phytoplankton community. Before incubation, part of the water sampled was treated with XAD-4 and XAD-8 resins to absorb dissolved organic compounds. Four dilutions were made by mixing untreated water with XAD-treated water in the ratios 100:0 (control), 70:30, 40:60 and 0:100. The phytoplankton specific growth rate increased significantly with the increased fraction treated with XAD in all but one incubation experiment. In these experiments, the specific growth rate was on average 9% higher in the fraction in which 100% was treated with XAD than in the controls. In the Rhine and Meuse river water, phytoplankton growth seemed to be inhibited by organic compounds. This inhibition was ascribed to the presence of dissolved organic micropollutants. Removing organic micropollutants using XAD resins to study the toxic effects of these compounds on field phytoplankton communities can be concluded to be a promising tool for risk assessment of micropollutants but needs to be supported by additional methodological research.     

Plant growth and reproduction along CO2 gradients: non-linear responses and implications for community change

The effects of rising atmospheric CO₂ concentrations on natural plant communities will depend upon the cumulative responses of plant growth and reproduction to gradual, incremental changes in climatic conditions. We analysed published studies of plant responses to elevated CO₂ to address whether reproductive and total biomass exhibit similar enhancement to elevated vs. ambient CO₂ concentrations, and to assess the patterns of plant response along gradients of CO₂ concentrations. In six annual plant species, mean enhancement at double ambient vs. ambient CO₂ was 1.13 for total biomass and 1.30 for reproductive biomass. The two measures were significantly correlated, but there was considerable scatter in the relationship, indicating that reproductive responses cannot be consistently predicted from enhancement of total biomass. Along experimental CO₂ gradients utilizing three concentrations, there was a great diversity of response patterns, including positive, negative, non-monotonic and non-significant (flat) responses. The distribution of response patterns differed for plants grown in stands compared to those grown individually. Positive responses were less frequent in competitive environments, and non-monotonic responses were more frequent. These results emphasize that interpolation of plant response based on enhancement ratios measured at elevated vs. ambient CO₂ concentrations is not sufficient to predict community responses to incremental changes in atmospheric conditions. The consequences of differential response patterns were assessed in a simulation of community dynamics for four species of annual plants. The model illustrates that the final community composition at a future point in time depends critically on both the magnitude and the rate of increase of atmospheric CO₂.     

Response of adult parasitoids ofBemisia tabaci (Hom.: Aleyrodidae) to leaf residues of selected cotton insecticides

The contact toxicity of eight insecticides to adults of four parasitoids of the sweetpotato whiteflyBemisia tabaci was evaluated in the laboratory. Two common Texas species,Eretmocerus sp. andEncarsia pergandiella Howard, and two exotic species,Eretmocerus mundus Mercet from Spain andEncarsia formosa Gahan from Greece were tested. Insecticides, applied as sprays to greenhouse-grown cotton plants at recommended rates were amitraz (Ovasyn^®), azinphosmethyl (Guthion^®), bifenthrin (Capture^®), buprofezin (Applaud^®), cypermethrin (Ammo^®), methyl parathion and thiodicarb (Larvin^®), with a water control. Parasitoid adults were confined on discs cut from leaves (1) sprayed the same day and (2) sprayed two days previously. Survival in both treatments was measured two and four days following exposure. Significant differences in toxicity were detected among the insecticides. Buprofezin was not toxic to any of the four parasitoids. When caged on leaves sprayed two days previously, only amitraz of the remaining compounds allowed significant general parasitoid survival after two days.E. mundus exhibited the greatest overall tolerance to insecticides, with 40% or more surviving 48 hr after confinement on leaves sprayed with amitraz, thiodicarb and cypermethrin. Survival was generally much reduced after 96 hr. In a separate test, fresh residues of endosulfan (Thiodan^®) were highly toxic at the two rates tested, but two day old residues at the lower rate allowed 76.7% survival ofE. mundus and 35% survival ofE. pergandiella after 48 hrs.     

Biology ofTrichogramma ostriniae (Hym.: trichogrammatidae) reared onOstrinia nubilalis (Lep.: pyralidae) and survey for additional hosts

Trichogramma ostriniae has been identified as a candidate for biological control ofOstrinia nubilalis. As little was known about the biology of this parasitoid when reared onO. nubilalis, we undertook experiments to quantify biological parameters important to mass-rearing and ase ofT. ostriniae. When reared continuously onO. nubilalis, femaleT. ostriniae on average lived 2.7 days and produced 24 progeny. Continuous access to honey resulted in a four-fold increase in longevity and fecundity and a significant increase in the percentage of females parasitizing eggs. Rates of fecundity and parasitism decreased with age of female. Likewise, emergence rates and percentage of female progeny decreased with age of parental female.T. ostriniae successfully parasitizedO. nubilalis eggs until the blackhead stage. Most parasitism of eggs and eclosion of adults occurred during the first half of photophase. Eggs of 13 Lepidopterans were parasitized byT. ostriniae. Eggs of the Noctuidae, Pyralidae, and Plutellidae experienced higher levels of parasitism than others tested.T. ostriniae appears to be similar to other species ofTrichogramma in several respects and does not possess any characteristics that limit its potential for mass rearing and use for augmentative biological control ofO. nubilalis.     

Interannual fluctuations in recruitment and growth of the sardine,Sardinops melanostictus,in the sea of Japan and adjacent waters

Recruitment and growth of the sardineSardinops melanostictus fluctuated markedly in the Sea of Japan and adjacent waters between 1978 and 1993. Stock size was calculated using Virtual Population Analysis and average body length in each age class was determined by the number of annual rings on the scales. There is an inverse correlation between average water temperature at a depth of 50 m in the coastal area of the mainland of Japan in winter (January to March) and recruitmentR defined as the number of individuals at 1 year old. There is also an inverse correlation between spawning stock sizeE and reproductive success in (R/E). A multiple regression model using spawning stock size and water temperature in winter as independent variables can explain 73% of variance in reproductive success. It suggests that both density-dependent and density-independent factors perform important roles determining reproductive success. There is an inverse correlation between body length and stock size and this suggests that there is a density-dependent effect on the growth of the sardine.