Analysis ofl-alanine:2-oxoglutarate aminotransferase isozymes in maize

Isozyme analysis ofl-alanine:2-oxoglutarate aminotransferase (ALT) in maize indicates that there are three genes encoding this enzyme activity. Two of the gene products interact with each other to form heterodimers, while the third gene product does not interact with the other two. Another isozyme that appears after gel electrophoresis and ALT staining is shown to be glutamate dehydrogenase-1. Anaerobic treatment does not result in increased ALT levels, indicating that the previously reported increase in alanine levels caused by this treatment may be due to increases in the level of pyruvate, a substrate of ALT.D. A. Russell was partially supported by a graduate student fellowship from the Division of Biology and Biomedical Sciences, Washington University. V. M. Peschke was partially supported by a postdoctoral fellowship from Monsanto. This research was supported by NIH Grant R01 GM34740.     

Multiple pyruvate decarboxylase genes in maize are induced by hypoxia

Two cDNA clones corresponding to anaerobically induced maize mRNAs were found to have homology to a previously identified maize pyruvate decarboxylase gene. DNA sequencing and RFLP mapping indicate that these cDNAs represent two additional maize pdc genes. Each of the clones is approximately 85% identical in predicted amino acid sequence to the other two. All three clones are induced by hypoxic stress, but with different levels and kinetics of induction.     

Dose-response curves for late functional changes in the normal rat brain after single carbon-on doses evaluated by magnetic resonance imaging: influence of follow-up time and calculation of relative biological effectiveness

This study investigated late effects in the brain after irradiation with carbon ions using a rat model. Thirty-six animals were irradiated stereotactically at the right frontal lobe using an extended Bragg peak with maximum doses between 15.2 and 29.2 Gy. Dose-response curves for late changes in the normal brain were measured using T1- and T2-weighted magnetic resonance imaging (MRI). Tolerance doses were calculated at several effect probability levels and times after irradiation. The MRI changes were progressive in time up to 17 months and remained stationary after that time. At 20 months the tolerance doses at the 50% effect probability level were 20.3 +/- 2.0 Gy and 22.6 +/- 2.0 Gy for changes in T1- and T2-weighted MRI, respectively. The relative biological effectiveness (RBE) was calculated on the basis of a previous animal study with photons. Using tolerance doses at the 50% effect probability level, RBE values of 1.95 +/- 0.20 and 1.88 +/- 0.18 were obtained for T1- and T2-weighted MRI. A comparison with data in the literature for the spinal cord yielded good agreement, indicating that the RBE values for single-dose irradiations of the brain and the spinal cord are the same within the experimental uncertainty.     

Dose-response curves and tolerance doses for late functional changes in the normal rat brain after stereotactic radiosurgery evaluated by magnetic resonance imaging: influence of end points and follow-up time

Late reaction of normal tissue is still a limiting factor in radiotherapy and radiosurgery of patients with brain tumors. Few quantitative data in terms of dose-response curves are available. In the present study, 99 animals were irradiated stereotactically at the right frontal lobe using a linear accelerator and single doses between 26 and 50 Gy. The diameter of the spherical dose distribution was 4.7 mm (80% isodose). Dose-response curves for late changes in the normal brain at 20 months were measured using T1- and T2-weighted magnetic resonance imaging (MRI). The dependence of the dose-response curves on the follow-up time and the definition of the biological end point were determined. Tolerance doses were calculated at several effect probability levels and times after irradiation. The MRI changes were found to be dependent on dose and progressive in time. At 20 months, the tolerance doses at a 50% effect probability level were 39.6 +/- 1.0 Gy and 42.4 +/- 1.4 Gy for changes in T1- and T2-weighted images, respectively. These dose-response curves can be used for further quantitative investigations on the influence of various treatment parameters, such as the application of charged particles, radiopharmaceuticals or the variation of tissue oxygenation.     

Measurement of hypoxia using the comet assay correlates with preirradiation microelectrode pO2 histography in R3327-AT rodent tumors

Polarographic determination of tumor oxygenation by Eppendorf histography is currently under investigation as a possible predictor of radiotherapy outcome. Alternatively, the alkaline comet assay has been proposed as a radiobiological approach for the detection of hypoxia in clinical tumor samples. Direct comparisons of these methods are scarce. One earlier study with different murine tumors could not establish a correlation, whereas a weak correlation was reported for a variety of human tumors. Considering the different end points and spatial resolution of the two methods, a direct comparison for a single tumor entity appeared desirable. Anaplastic R3327-AT Dunning prostate tumors were grown on Copenhagen rats to volumes of 1-6 cm(3). Eppendorf histography (100-200 readings in 5 parallel tracks) for 8 different tumors revealed various degrees of oxygenation, with median pO(2) values ranging from 1.1 to 23 mmHg. Within 5 min after an acute exposure to 8 Gy (60)Co gamma rays, tumors were excised from killed animals and rapidly cooled to limit repair, and a single cell suspension was prepared for use with the comet assay. The resulting comet moment distributions did not exhibit two subpopulations (one hypoxic and the other aerobic), and a hypoxic fraction could not be calculated. Instead, the average comet moment distribution was taken as a parameter of overall strand break induction. Corresponding experiments with tumor cells grown in vitro allowed us to derive the relationship between the oxygen enhancement ratio (OER) for the average comet moment and oxygen partial pressure (Howard-Flanders and Alper formula). The validity of this relationship was inferred for cells exposed in situ, and the convolution of a pO(2) distribution with the formula of Howard-Flanders and Alper yielded an array of expected OER values for each tumor. The average expected OER correlated well with the average comet moment (r = 0.89, P < 0.01), and the in situ comet moment distributions could be predicted from the Eppendorf data when 50% repair was taken into account, assuming a 5-min damage half-life. The findings confirm the potential of interstitial polarography to reflect radiobiologically relevant intracellular oxygenation, but also underscore the confounding influence of differences in repair that may occur when cells are prepared from irradiated tissues for use with the comet assay.     

Design, synthesis and biological activity of novel peptidyl benzyl ketone FVIIa inhibitors

Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formulations of rFVIIa, which is a serine protease used for the treatment of hemophilia A and B inhibitor patients. A library of compounds was synthesized with different tripeptide sequences, N-terminals and d-amino acids in the P3 position. Cbz-d-Phe-Phe-Arg-bk (33) was found to be the best candidate with a potency of K_i = 8 μM and no substantial inhibition of related blood coagulation factors (thrombin and FXa). Computational studies revealed that 33 has a very stable binding conformation due to intramolecular hydrogen bonds, which cannot be formed with l-Phe in the P3 position. Nonpolar amino acids were found to be superior, probably due to a minimization of the cost of desolvation upon binding to FVIIa.     

Interaction of liquid epicuticular hydrocarbons and tarsal adhesive secretion in Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)

Species of various insect orders possess specialised tarsal adhesive structures covered by a thin liquid film, which is deposited in the form of footprints. This adhesive liquid has been suggested to be chemically and physiologically related to the epicuticular lipid layer, which naturally covers the body of insects and acts as the prime barrier to environmental stresses, such as desiccation. The functional efficiency of the layer, however, is jeopardised by partial melting that may occur at physiological temperatures. In this study, light microscopic images of elytral prints show that the epicuticular lipid layer of the Colorado potato beetle Leptinotarsa decemlineata actually is partially liquid and chemical investigations reveal the high similarity of the epicuticular hydrocarbon pattern and the tarsal liquid. By means of chemical manipulation of the surface hydrocarbon composition of live beetles, the substance exchange between their tarsal adhesive hairs and the body surface is monitored. Histological sections of L. decemlineata tarsi, furthermore, reveal glandular cells connected to individual adhesive setae and departing from these results, an idea of a general mechanism of tarsal secretion is developed and discussed in a functional–ecological context.     

Genome-wide analysis of light-dependent transcript accumulation patterns during early stages of Arabidopsis seedling deetiolation

Light is among the most important exogenous factors that regulate plant development. To sense light quality, intensity, direction, and duration, plants have evolved multiple photoreceptors that enable the detection of photons from the ultraviolet B (UV-B) to the far-red spectrum. To study the effect of different light qualities on early gene expression, dark-grown Arabidopsis (Arabidopsis thaliana) seedlings were either irradiated with continuous far-red, red, or blue light or received pulses of red, UV-A, or UV-A/B light. The expression profiles of seedlings harvested at 45 min and 4 h were determined on a full genome level and compared with the profiles of dark controls. Data were used to identify light-regulated genes and to group these genes according to their light responses. While most of the genes were regulated by more than one light quality, a considerable number of UV-B-specific gene expression responses were obtained. An extraordinarily high similarity in gene expression patterns was obtained for samples that perceived continuous irradiation with either far-red or blue light for 4 h. Mutant analyses hint that this coincidence is caused by a convergence of the signaling cascades that regulate gene expression downstream of cryptochrome blue light photoreceptors and phytochrome A. Whereas many early light-regulated genes exhibited uniform responses to all applied light treatments, highly divergent expression patterns developed at 4 h. These data clearly indicate that light signaling during early deetiolation undergoes a switch from a rapid, but unspecific, response mode to regulatory systems that measure the spectral composition and duration of incident light.