Europium(III) luminescence and tyrosine to terbium(III) energy-transfer studies of invertebrate (octopus) calmodulin.

The effects of minor differences in the amino acid sequences between a vertebrate (bovine testes) and an invertebrate (octopus) calmodulin on metal ion binding were investigated via laser-induced Eu3+ and Tb3+ luminescence. Amino acid substitutions at residues which are coordinated to the metal ion do not produce any detectable changes in the 7F0----5D0 excitation spectrum of the Eu3+ ion bound to octopus calmodulin relative to bovine testes calmodulin; only minor differences in the excited-state lifetime values in D2O solution are observed. The dissociation constants for Eu3+ (1.0 +/- 0.2 microM) and Tb3+ (5 +/- 1 microM) from the weak lanthanide binding sites (III and IV, numbered from the amino terminus) of octopus calmodulin were measured using luminescence techniques. Both values agree well with those reported previously for bovine testes calmodulin [Mulqueen, P. M., Tingey, J. M., & Horrocks, W. D., Jr. (1985) Biochemistry 24, 6639-6645]. The measured dissociation constant of Eu3+ bound in the tight lanthanide binding sites (I and II) is 6 +/- 2 nM for octopus calmodulin and 12 +/- 2 nM for bovine testes calmodulin. The distances between sites I and II (12.4 +/- 0.5 A) and sites III and IV (11.7 +/- 0.8 A) were determined from F?rster-type energy transfer in D2O solutions of octopus calmodulin containing bound Eu3+ donor and Nd3+ acceptor ions. F?rster theory parameters for nonradiative energy transfer between Tyr138 and Tb3+ ions bound at sites III and IV of octopus calmodulin were comprehensively evaluated, including a dynamics simulation of the orientation factor kappa 2. This theory is found to account quantitatively for the observed energy-transfer efficiency as evaluated from the observed sensitized Tb3+ emission.     

Functional analysis of a complex oncogene arrangement in biotype III Agrobacterium tumefaciens strains

The ubiquitous grapevine-associated octopine/cucumopine Ti plasmids of biotype III Agrobacterium tumefaciens strains carry two T regions, TA and TB, with a complex oncogene arrangement. Within the octopine/cucumopine group, two main strain types were identified: large TA strains with a TA region resembling the TL region of the biotype I octopine strain Ach5 and small TA strains with a similar T region organization as the large TA strains but with a large internal TA deletion. Structural and functional studies of the representative large TA strain Tm4 revealed six oncogenes. Each oncogene was inserted in a disarmed vector and tested for biological activity using the corresponding oncogenes of Ach5 as standards. Five Tm4 oncogenes, TA-iaaM, T-ipt, T-6b, TB-iaaH and TB-iaaM, were shown to be active, the IS-interrupted TA-iaaH gene was inactive. To study the role of each gene in the pTiTm4 context, several single and multiple pTiTm4 mutations were constructed. It was shown that whereas TA-iaaM and TB-iaaH are essential for tumour formation on grapevine, T-ipt, T-6b and TB-iaaM are not. The avirulence of the TA-iaaM ^- mutant was shown to be due to an inhibitory effect of the T-ipt gene, since a TA-iaaM ^- /T-ipt ^- double mutant was fully virulent. We conclude that the TA-iaaM gene of large TA strains is specifically required to counteract the tumour growth inhibiting activity of the T-ipt gene. Both TA-iaaM and T-ipt are absent from the small TA strains. A model on the roles and interactions of the different oncogenes in large TA and small TA strains is presented.     

Oxygen transport in the blood of arctic mammals: adaptation to local heterothermia

Summary The oxygen binding of whole blood from humans and two arctic mammals, reindeer and muskox, has been studied as a function of carbon dioxide and temperature. All bloods display a marked Bohr effect with Bohr coefficients in the range –0.44––0.73. The Bohr effect is more pronounced at 20°C. The temperature sensitivity of reindeer and muskox blood expressed by the apparent heat of oxygenation, H, is almost three times lower than that of human HbA under the same experimental conditions. This thermodynamic difference gives special benefits to arctic mammals with large heterothermy by safeguarding oxygen unloading at very low ambient temperatures.     

Preference for plants damaged by conspecific larvae in ovipositing cabbage root flies: influence of stimuli from leaf surface and roots

In laboratory dual-choice assays females of the cabbage root fly, Delia radicum, prefer for oviposition plants with roots damaged by conspecific larvae to undamaged controls. Cauliflower and kale plants were inoculated with root fly eggs (25 per plant) and the hatching larvae were allowed to feed on the roots for various periods of time (1–17 days). After 4 (cauliflower) or 5 (kale) days of larval feeding the oviposition preference was most pronounced and flies laid between 64% and 68% of their eggs near plants with damaged roots. Later, with increasing damage but fewer surviving, and thus actively feeding, larvae, the magnitude of the preference declined. The preference for plants already damaged by conspecific larvae may contribute to the previously observed aggregated distribution of D. radicum eggs in Brassica crop fields.Further experiments revealed that the sensory cues inducing this oviposition preference originate from the complex consisting of the damaged roots, the surrounding substrate (soil) and associated microbes, rather than from the aerial plant parts. In choice assays using the root-substrate complex of damaged and control plants (aerial parts removed), the observed preference for damaged roots was similar to that found for the entire plant but was more pronounced. The damaged roots alone, compared to control roots, received up to 72% (cauliflower) and 75% (kale) of the eggs. By contrast, surrogate leaves sprayed with methanolic leaf surface extracts from the most preferred plants which had been damaged were not discriminated from surrogate leaved sprayed with extracts of the respective control plants. Analysis of glucosinolate levels in methanolic leaf surface extracts revealed that root damage resulted in enhanced concentrations of indole-glucosinolates on the leaf surface in kale but not in cauliflower. Although indole-glucosinolates are oviposition stimulants for the cabbage root fly, the induced changes were apparently too small to influence oviposition behaviour.     

NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces: Cyprinidae)

Chromosomal polymorphism regarding number of NOR sites in the cyprinid fish Chondrostoma lusitanicum was examined using C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ (CMA₃). The analysis of heterochromatic regions allowed a more precise identification of the centromeric regions and the proposal of a revised haploid chromosome formula (7M: 15S: 3A). We describe variability in the number of NOR regions per genome, number of active NOR sites per cell, and relative size of individual NORs. Individuals expressed two or four NOR-bearing chromosomes. Polymorphism was detected in all the populations studied and sex-related differences were not found. The observed chromosomal NOR phenotypes suggest the occurrence of structural rearrangements during the evolutionary process of this diploid leuciscine cyprinid.     

Effect of Protease Inhibitors on Early Events of Apoptosis

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization ofin situDNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence ofin situDNA strand breaks. DNA stability was estimated by the measure of its sensitivityin situto denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of ≥50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNAin situ,and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to ≥50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.     

Linkage disequilibrium and extended haplotypes in the HLA-A to D6S105 region: implications for mapping the hemochromatosis gene (HFE)

The hemochromatosis gene (HFE) maps to 6p21.3, in close linkage with the HLA Class I genes. Linkage disequilibrium (LD) studies were designed to narrow down the most likely candidate region for HFE, as an alternative to traditional linkage analysis. However, both the HLA-A and D6S105 subregions, which are situated 2–3 cM and approximately 3 Mb apart, have been suggested to contain HFE. The present report extends our previous study based upon the analysis of a large number of HFE and normal chromosomes from 66families of Breton ancestry. In addition to the previously used RFLP markers spanning the 400-kb surrounding HLA-A, we examined three microsatellites: D6S510, HLA-F, and D6S105. Our combined data not only confirm a peak of LD at D6S105, but also reveal a complex pattern of LD over the i82 to D6S105 interval. Within our ethnically well-defined population of Brittany, the association of HFE with D6S105 is as great as that with HLA-A, while the internal markers display a lower LD. Fine haplotype analysis enabled us to identify two categories of haplotypes segregating with HFE. In contrast to the vast majority of normal haplotypes, 50% of HFE haplotypes are completely conserved over the HLA-A to D6S105 interval. These haplotypes could have been conserved through recombination suppression, selective forces and/or other evolutionary factors. This particular haplotypic configuration might account for the apparent inconsistencies between genetic linkage and LD data, and additionally greatly complicates positional cloning of HFE through disequilibrium mapping.The authors contributed equally to this work     

Ecophysiology and distribution of the endemic leafless spurgeEuphorbia aphylla and the introducedE. tirucalli (Euphorbiaceae,Euphorbia sect.Tirucalli) in the Canary Islands

The leafless spurgeEuphorbia aphylla (Euphorbiaceae), an endemic species restricted to several of the Canary Islands where it inhabits coastal and arid localities, expresses Crassulacean Acid Metabolism (CAM) when it is subjected to summer drought. A flexible CAM is consistent with the general ecology of the species. It is the only member of sect.Tirucalli native to the Canary Islands and is at the north-western edge of the section's biogeographical range. The other members of the section have a paleotropic distribution and are found throughout Africa. Many of them are regarded as obligate CAM plants, includingE. tirucalli which was used as a comparison in ecophysiological experiments examining the response ofE. aphylla to drought and temperature.