Iodoazomycin riboside (1-(5'-iodo-5'-deoxyribofuranosyl)-2-nitroimidazole), a hypoxic cell marker. I. Synthesis and in vitro characterization

Misonidazole (MISO), a selective radiosensitizer of hypoxic cells, forms adducts with cellular biomolecules with rates which are 30-50 X higher under hypoxic as compared to aerobic conditions of incubation. This technique of sensitizer adduct formation was proposed as a possible means of measuring the hypoxic fraction of solid tumors by noninvasive procedures. Iodoazomycin riboside (5'-IAZR) and 5'-[125I]AZR were synthesized and chemically characterized. Measurements of in vitro cytotoxicity and radiosensitizing ability with EMT-6 tumor cells in vitro indicated that 5'-IAZR is approximately 3 X more toxic and effective than is azomycin riboside (AZR) and approximately 10 X more toxic and effective than is MISO. 5'-[125I]AZR was shown to selectively bind to hypoxic EMT-6 cells at rates which were 2.5-3 X faster than those of MISO. The absolute rates of binding of 5'-IAZR to hypoxic cells at concentrations of 10-100 microM are the highest observed in this laboratory for any hypoxic cell radiosensitizer tested to date. These data suggest that 5'-IAZR, when labeled with an appropriate radioisotope (e.g., 131I), might be a useful marker for hypoxic cells in solid tumors amenable to noninvasive detection. Additional studies with animal tumor models appear to be warranted.     

Incorporation of fucose in the intact heart and dissociated heart cells of the chick embryo

The incorporation of [³H]fucose into cell-bound and medium-released TCA-precipitable fractions was determined in intact hearts and dissociated heart cells of the 4-day chick embryo. The amount of released label was found to be much greater in the dissociated cells than in intact hearts both in absolute quantities and in proportion to cell-bound label.     

Substrate-enzyme interactions and catalytic mechanism in phospholipase C: a molecular modeling study using the GRID program.

Based on the high-resolution X-ray crystallographic structure of phospholipase C from Bacillus cereus, the orientation of the phosphatidylcholine substrate in the active site of the enzyme is proposed. The proposal is based on extensive calculations using the GRID program and molecular mechanics geometry relaxations. The substrate model has been constructed by successively placing phosphate, choline and diacylglycerol moieties in the positions indicated from GRID calculations. On the basis of the resulting orientation of a complete phosphatidylcholine molecule, we propose a mechanism for the hydrolysis of the substrate.     

Mechanical and metabolic determinants of the preferred step width in human walking

We studied the selection of preferred step width in human walking by measuring mechanical and metabolic costs as a function of experimentally manipulated step width (0.00-0.45L, as a fraction of leg length L). We estimated mechanical costs from individual limb external mechanical work and metabolic costs using open circuit respirometry. The mechanical and metabolic costs both increased substantially (54 and 45%, respectively) for widths greater than the preferred value (0.15-0.45L) and with step width squared (R(2) = 0.91 and 0.83, respectively). As predicted by a three-dimensional model of walking mechanics, the increases in these costs appear to be a result of the mechanical work required for redirecting the centre of mass velocity during the transition between single stance phases (step-to-step transition costs). The metabolic cost for steps narrower than preferred (0.10-0.00L) increased by 8%, which was probably as a result of the added cost of moving the swing leg laterally in order to avoid the stance leg (lateral limb swing cost). Trade-offs between the step-to-step transition and lateral limb swing costs resulted in a minimum metabolic cost at a step width of 0.12L, which is not significantly different from foot width (0.11L) or the preferred step width (0.13L). Humans appear to prefer a step width that minimizes metabolic cost.     

Large differences in erythrocyte stability between species reflect different antioxidative defense mechanisms

We have developed a screening assay for erythrocyte stability, which is rapid, easy, inexpensive, robust, and suitable for handling a large number of samples in parallel. Erythrocytes are incubated overnight in 96-well microtiter plates in absence or presence of various oxidants, intact cells are pelleted by centrifugation, and lysis is determined by release of intracellular constituents into the supernatant as either activity of lactate dehydrogenase (LDH) or absorbance of hemoglobin at 406 nm. There is good correlation between the methods. A number of advantages by the present method are that only small amounts of blood is needed, washing is optional, erythrocytes may be stored for at least one day before assay, and large numbers of samples can be handled in parallel. Using this set-up, we have compared erythrocyte stability from several different animal species. We find that erythrocyte susceptibility towards lysis induced by H₂O₂ and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is highly species dependent. The different susceptibility between species is due to cellular components, since swapping of plasma between species has little or no effect. As a novel observation, we find that erythrocytes from chicken are the most sensitive of the species tested towards lysis by H₂O₂ and are almost four orders of magnitude more sensitive than erythrocytes from man. This is due to a much lower content of catalase in erythrocytes from chicken. A more narrow range is observed for susceptibility towards AAPH and the ranking between the species is different. Thus, chicken erythrocytes are more resistant towards AAPH than some mammals by up to two orders of magnitude. This differential stability towards different oxidative stressors is likely due to evolution/selection of different defense mechanisms.     

A stretch of positively charged amino acids at the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into the peroxisomal membrane

Pex3p is a peroxisomal membrane protein that is essential for peroxisome biogenesis. Here, we show that a conserved stretch of positively charged amino acids (Arg(11)-X-Lys-Lys-Lys(15)) in the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into its target membrane. Despite the strong conservation, this sequence shows a high degree of redundancy. Substitution of either Arg(11), Lys(13), Lys(14), or Lys(15) with uncharged or negatively charged amino acids did not interfere with Pex3p location and function. However, a mutant Pex3p, carrying negatively charged amino acids at position 13 and 15 (K13E/K15E), caused moderate but significant defects in peroxisome assembly and matrix protein import. Additional changes in the N terminus of Pex3p, e.g. replacing three or four of the positively charged amino acids with negatively charged ones, led to a typical pex3 phenotype, i.e. accumulation of peroxisomal matrix proteins in the cytosol and absence of peroxisomal remnants. Also, in these cases, the mutant Pex3p levels were reduced. Remarkably, mutant Pex3p proteins were mislocalized to mitochondria or the cytosol, depending on the nature of the mutation. Furthermore, in case of reduced amounts of Pex3p, the levels of other peroxisomal membrane proteins, e.g. Pex10p and Pex14p, were also diminished, suggesting that Pex3p maybe involved in the recruitment or stabilization of these proteins (in the membrane).     

Analysis of mannose selection used for transformation of sugar beet

Various factors affecting mannose selection for the production of transgenic plants were studied using Agrobacterium tumefaciens-mediated transformation of sugar beet (Beta vulgaris L.) cotyledonary explants. The selection system is based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable gene and mannose as selective agent. Transformation frequencies were about 10-fold higher than for kanamycin selection but were only obtained at low selection pressures (1.0–1.5 g/l mannose) where 20–30% of the explants produced shoots. The non-transgenic shoots were eliminated during the selection procedure by a stepwise increase in the mannose concentration up to 10 g/l. Analysis of the transformed shoots showed that the PMI activity varied from 2.4 mU/mg to 350 mU/mg but the expression level was independent of the selection pressure. Complete resistance to mannose of transformed shoots was observed already at low PMI activities (7.5 mU/mg). Genomic DNA blot analysis confirmed the presence of the PMI gene in all transformants analysed. The possible mode of action of mannose selection compared to other selection methods is discussed.     

Decline in measles mortality: nutrition,age at infection,or exposure?

The mortality from measles was studied in an urban area of Guinea-Bissau one year before and five years after the introduction of a vaccination programme. The years after the introduction of immunisation saw a decline in mortality among unvaccinated children with measles. This decline occurred despite a lower age at infection and an increasing prevalence of malnourished children. State of nutrition (weight for age) did not affect the outcome of measles infection. The incidence of isolated cases, however, increased in the period after the introduction of measles vaccination. As mortality was lower among these cases, diminished clustering explained some of the reduction in mortality. Comparison between the urban district and a rural area inhabited by the same ethnic group showed a lower age at infection, less clustering of cases, and lower case fatality ratios in the urban area.Endemic transmission of measles in urban districts leads to less clustering of cases, which may help explain the usually lower case fatality ratios in these areas. As measles vaccination increases herd immunity and diminishes clustering of cases, it may reduce mortality even among unvaccinated children who contract the disease.     

Large-scale bioreactor production of the herbicide-degrading Aminobacter sp. strain MSH1

The Aminobacter sp. strain MSH1 has potential for pesticide bioremediation because it degrades the herbicide metabolite 2,6-dichlorobenzamide (BAM). Production of the BAM-degrading bacterium using aerobic bioreactor fermentation was investigated. A mineral salt medium limited for carbon and with an element composition similar to the strain was generated. The optimal pH and temperature for strain growth were determined using shaker flasks and verified in bioreactors. Glucose, fructose, and glycerol were suitable carbon sources for MSH1 (μ?=?0.1 h^?1); slower growth was observed on succinate and acetic acid (μ?=?0.01 h^?1). Standard conditions for growth of the MSH1 strain were defined at pH 7 and 25 °C, with glucose as the carbon source. In bioreactors (1 and 5 L), the specific growth rate of MSH1 increased from μ?=?0.1 h^?1 on traditional mineral salt medium to μ?=?0.18 h^?1 on the optimized mineral salt medium. The biomass yield under standard conditions was 0.47 g dry weight biomass/g glucose consumed. An investigation of the catabolic capacity of MSH1 cells harvested in exponential and stationary growth phases showed a degradation activity per cell of about 3?×?10^?9 μg BAM h^?1. Thus, fast, efficient, large-scale production of herbicide-degrading Aminobacter was possible, bringing the use of this bacterium in bioaugmentation field remediation closer to reality.