Immunogold Localisation of the Intermediate Chain within the Protein Complex of Cytoplasmic Dynein

It was our goal to determine the location of the intermediate chain within the complex of cytoplasmic dynein by immunoelectron microscopy. To do so we generated two monoclonal antibodies (m74-1 and m74-2) specific for the intermediate chain. Both antibodies recognised the intermediate chain by sodium dodecyl sulphate–polyacrylamide gel electrophoresis immunoblot and ELISA assays of native and denatured proteins. When sucrose density gradient-purified cytoplasmic dynein from bovine brain was incubated with the gold-conjugated monoclonal antibodies, m74-1 and m74-2, and examined by negative staining, the gold label was found opposite the globular heads at the base of the V-shaped stalk of the motor complex. The labelling of the intermediate chain is the first mapping of a component within cytoplasmic dynein. The identification of the intermediate chain at the base of the complex supports a possible docking function of the intermediate chain.     

Global vegetation models: incorporating transient changes to structure and composition

Abstract. We describe an approach for developing a Dynamic Global Vegetation Model (DGVM) that accounts for transient changes in vegetation distribution over a decadal time scale. The DGVM structure is based on a linkage between an equilibrium global vegetation model and smaller scale ecosystem dynamics modules that simulate the rate of vegetation change. Vegetation change is classified into four basic types, based largely on the projected change in above-ground biomass of the vegetation. These four types of change are: (1) dieback of forest, shrubland or grassland; (2) successional replacement within forest, shrubland or grassland; (3) invasion of forest, shrubland or grassland; (4) change in tree/grass ratio. We then propose an approach in which the appropriate ecosystem dynamics module for each type of change is applied and the grid cells of the global model updated accordingly. An approach for accounting for fire, as an example of a disturbance which may strongly influence the rate and spatial pattern of forest dieback, is incorporated. We also discuss data needs for the development, calibration and validation of the model.     

DNA damage and repair in somatic and germ cells in vivo

Alkylation-induced germ cell mutagenesis in the mouse versus Drosophila is compared based on data from forward mutation assays (specific-locus tests in the mouse and in Drosophila and multiple-locus assays in the latter species) but not including assays for structural chromosome aberrations. To facilitate comparisons between mouse and Drosophila, forward mutation test results have been grouped into three categories. Representatives of the first category are MMS (methyl methanesulfonate) and EO (ethylene oxide), alkylating agents with a high s value which predominantly react with ring nitrogens in DNA. ENU (N-ethyl-N-nitrosourea), MNU (N-methyl-N-nitrosourea), PRC (procarbazine), DEN (N-nitrosodiethylamine), and DMN (N-nitrosodimethylamine) belong to the second category. These agents have in common a considerable ability for modification at oxygens in DNA. Cross-linking agents (melphalan, chlorambucil, hexamethylphosphoramide) from the third category.The most unexpected, but encouraging outcome of this study is the identification of common features for three vastly different experimental indicators of genotoxicity: hereditary damage in Drosophila males, genetic damage in male mice, and tumors (TD₅₀ estimates) in rodents. Based on the above three category classification scheme the following tentative conclusions are drawn. Monofunctional agents belonging to category 1, typified by MMS and EO, display genotoxic effects in male germ cell stages that have passed meiotic division. This phenomenon seems to be the consequence of a repair deficiency during spermiogenesis for a period of 3–4 days in Drosophila and 14 days in the mouse. We suggest that the reason for the high resistance of premeiotic stages, and the generally high TD₅₀ estimates observed for this class in rodents, is the efficient error-free repair of N-alkylation damage. If we accept this hypothesis, then the increased carcinogenic potential in rodents, seen when comparing category 2 (ENU-type mutagens) to category 1 (MMS-type mutagens), along with the ability of category 2 genotoxins to induce genetic damage in premeiotic stages, must presumably be due to their enhanced ability for alkylations at oxygens in DNA; it is this property that actually distinguishes the two groups from each other. In contrast to category 1, examination of class 2 genotoxins (ENU and DEN) in premeiotic cells of Drosophila gave no indication for a significant role of germinal selection, and also removal by DNA repair was less dramatic compared to MMS. Thus category 2 mutagens are expected to display activity in a wide range of both post- and premeiotic germ cell stages. A number of these agents have been demonstrated to be among the most potent carcinogens in rodents. In terms of both hereditary damage and the initiation of cancers (low TD₅₀), cross-linking agents (category 3) comprise a considerable genotoxic hazard. Doubling doses for the mouse SLT have been determined for four cross-linking agents not requiring metabolic conversion and in all four cases the doubling doses for these agents were lower than those for MMS, DES and EMS. In support of this conclusion, two of 10 genotoxic agents, for which data on chromosomal aberrations were available for both somatic cells and germ cells in mice, were cross-linking agents and again the doubling dose estimates are lower than for monofunctional agents. Four cross-linking agents induced mutations in stem cell spermatogonia indicating that this type of agent can be active in a wide range of germ cell stages.Quite in contrast to what is generally observed in unicellular systems and in mammalian cells in culture, both cross-linking agents and MMS-type mutagens (high s value) predominantly produce deletion mutations in postmeiotic male germ cell stages. This is the uniform picture found for both Drosophila and the mouse. It is concluded that in vitro systems, in contrast to Drosophila germ cells, fail to predict this very intriguing feature of mouse germ line mutagenesis. In addition to their potential for induction of deletions and other rearrangements, cross-linking agents are among the most efficient inducers of mitotic recombination in Drosophila. Thus there are several mechanisms by which cross-linking agents may cause loss of heterozygosity for long stretches of DNA sequences, leading to expression of recessive genes. Since a substantial portion of agents used in the chemotherapy of cancers have cross-linking potential, the potential hazards of hereditary damage and cancers associated with this class of genotoxins should, in our opinion, receive more attention than they have in the past.     

Increased chilling tolerance and altered carbon metabolism in tomato leaves following application of mechanical stress

We investigated the effects of brushing on the chilling tolerance and metabolism of nonstructural carbohydrates (soluble sugars and starch) in tomato leaves before, during and after a chilling stress. Tomato plants ( Lycopersicon esculentum Mill. cv. Caruso) were cultivated either without mechanical stress application (control plants) or with daily brushing treatments for 15 days (brushed plants), prior to a 7-day chilling treatment (8/5°C day/night). Brushing resulted in shorter plants with a 34% reduction in leaf dry weight per area and a 59% reduction of soluble sugars and starch, on a dry weight basis. The sugar to starch ratio was not affected by brushing. A greater chilling tolerance in the brushed plants was demonstrated by the maintenance of a significantly higher PSII efficiency in brushed plants (42%) compared to that of the control plants (30%) after 7 days of chilling treatment, less visible damage to the leaf tissue, and a more rapid resumption of growth during 3 days of recovery as compared to control plants. During the chilling treatment levels of soluble sugars per leaf dry weight increased 15-fold in the brushed plants and 5-fold in control plants. In the present study we have demonstrated that brushing can increase chilling tolerance in tomato plants. The observed differences in chilling tolerance and concentration of soluble sugars in the leaves may indicate an involvement of soluble sugar levels in acclimation to chilling.     

Novel nuclear ribonucleoprotein structural components in the dormouse adrenal cortex during hibernation

Adrenocortical cell nuclei of the dormouse Muscardinus avellanarius were investigated by electron microscopic immunocytochemistry in hibernating, arousing and euthermic individuals. While the basic structural constituents of the cell nucleus did not significantly were found in nuclei of hibernating dormice. Lattice-like bodies (LBs), clustered granules (CGs), fibrogranular material (FGM) and granules associated with bundles of nucleoplasmic fibrils (NF) all contained ribonucleoproteins (RNPs), as shown by labeling with anti-snRNP (small nuclear RNP), anti-m₃G-capped RNA and anti-hnRNP (heterogeneous nuclear RNP) antibodies. Moreover, the FGM also showed immunoreactivity for the proliferation associated nuclear antigen (PANA) and the non-snRNP splicing factor SC-35. All these nuclear structural components disappeared early during arousal and were not found in euthermic animals. These novel RNP-containing structures, which have not been observed in other tissues investigated so far in the same animal model, could represent storage and/or processing sites for pre-mRNA during the extreme metabolic condition of hibernation, to be quickly released upon arousal. NFs, which had been sometimes found devoid of associated granules in nuclei of brown adipose tissue from hibernating dormice, were present in much higher amouts in adrenocortical cell nuclei; they do not contain RNPs and their role remains to be elucidated. The possible roles of these structures are discussed in the frame of current knowledge of morpho-functional relationships in the cell nucleus.     

The Cross-Linking Agent Hexamethylphosphoramide Predominantly Induces Intra-Locus and Multi-Locus Deletions in Postmeiotic Germ Cells of Drosophila

The nature of DNA sequence changes induced by the cross-linking agent hexamethylphosphoramide (HMPA) within and in the vicinity of the vermilion locus of Drosophila melanogaster that produce a vermilion mutant phenotype was analyzed after exposure of postmeiotic male germ cells. Mutagenized males were mated to either females wild-type (exr(+)) for nucleotide excision repair (NER) or to females having a deficiency (exr(-)) for NER. Rearrangements, mostly deletions, represented by far the most frequent type of mutational events induced by HMPA that are detected as vermilion mutations. In the exr(+) group, all but one (a double substitution) of 21 mutants characterized were large sequence changes: we found 5 intra-locus deletions, 3 intra-locus deletions associated with insertions and 12 multi-locus deletions. When taken together, deletions and deletion/insertion mutations represent 96% of the HMPA-induced DNA modifications obtained under proficient repair conditions. Of the 10 mutants obtained from crosses with exr(-) females, 6 intra-locus and 2 multi-locus deletions were found, as opposed to just 1 point mutation and 1 double substitution. The ``hypomutability effect' observed with exr(-) genotypes in relation to the wild type seems to be caused by a decrease in the frequency of multi-locus deletions in the former group. The results suggest that the NER system is involved in the generation of multi-locus deletions, whereas intra-locus deletions appear to be formed through a postreplication slipped-misrepair pathway. It is concluded that an eukaryotic in vivo system with no limitations for the recovery of multi-locus deletions, such as vermilion, should be used for the analysis of DNA damage induced by cross-linking agents.     

BBReader: A computer program for the combined use of the BioMagResBank and PDB databases

A computer program (BBReader) was developed which performs an inverse search in theBioMagResBank database. Given (cross) peak positions of a protein, the program searchesfor atoms with matching chemical shifts and suggests possible assignments for user-specifiedhomo- and heteronuclear one- to three-dimensional COSY- and NOESY-type experiments.It can handle 1H, 13C and 15N spectra. Distance information from PDB files can be utilizedfor filtering possible NOESY cross peak assignments.