PROBLEMS WITH TESTING INBREEDING AVOIDANCE: THE CASE OF THE COLLARED FLYCATCHER

Four year's data on collared flycatchers, Ficedula albicollis, breeding in a nestbox plot on the island of Gotland, Sweden, was used to investigate whether individuals avoid mating with close kin (i.e., parents or sibs). Only one case of close inbreeding (0.5% of all pairs) was observed during the years of study. The observed frequency of close inbreeding was compared to expected frequencies based on two different null models. Assuming no inbreeding avoidance behaviors (e.g., dispersal or kin recognition), but taking into account the fact that mortality, and different arrival and pairing times of individuals reduce the probability of mating with close kin, the expected frequency of close inbreeding is 10% and 15% for female and male recruits (i.e., born in the study plot), respectively. However, assuming mating to be random within the study plot reduced the expected frequency of close inbreeding to 1% or less for both males and females. Consequently, conclusions drawn concerning inbreeding avoidance depend on the null model used. Contrasting estimated costs of tolerating close inbreeding with those of avoiding it (by dispersal to other plots), however, suggests that the costs of avoiding close inbreeding are substantially greater than those of tolerating it. Therefore, although inbreeding avoidance cannot be rejected as a cause of dispersal of this species, it is not the primary cause, and particularly not for sex-biased dispersal. The general problems of investigating inbreeding avoidance are discussed. It is argued that all previous null models based on random mating in finite populations produce expected frequencies of close inbreeding that in fact include inbreeding avoidance, since they implicitly assume random dispersal within a finite population. Thus, comparisons between observed and expected frequencies of close inbreeding based on random mating are inadequate. The most promising method of investigating inbreeding avoidance is to experimentally study individual movements and mating preferences in the presence and absence of close kin.     

Bacteriochlorophyll organization and energy transfer kinetics in chlorosomes from Chloroflexus aurantiacus depend on the light regime during growth

We have used measurements of fluorescence and circular dichroism (CD) to compare chlorosome-membrane preparations derived from the green filamentous bacterium Chloroflexus aurantiacus grown in continuous culture at two different light-intensities. The cells grown under low light (6 mol m^–2 s^–1) had a higher ratio of bacteriochlorophyll (BChl) c to BChl a than cells grown at a tenfold higher light intensity; the high-light-grown cells had much more carotenoid per bacteriochlorophyll.The anisotropy of the Q_Y band of BChl c was calculated from steady-state fluorescence excitation and emission spectra with polarized light. The results showed that the BChl c in the chlorosomes derived from cells grown under high light has a higher structural order than BChl c in chlorosomes from low-light-grown cells. In the central part of the BChl c fluorescence emission band, the average angles between the transition dipole moments for BChl c molecules and the symmetry axis of the chlorosome rod element were estimated as 25° and 17° in chlorosomes obtained from the low- and high-light-grown cells, respectively.This difference in BChl organization was confirmed by the decay associated spectra of the two samples obtained using picosecond single-photon-counting experiments and global analysis of the fluorescence decays. The shortest decay component obtained, which probably represents energy-transfer from the chlorosome bacteriochlorophylls to the BChl a in the baseplate, was 15 ps in the chlorosomes from high-light-grown cell but only 7 ps in the preparation from low-light grown cells. The CD spectra of the two preparations were very different: chlorosomes from low-light-grown cells had a type II spectrum, while those from high-light-grown cells was of type I (Griebenow et al. (1991) Biochim Biophys Acta 1058: 194–202). The different shapes of the CD spectra confirm the existence of a qualitatively different organization of the BChl c in the two types of chlorosome.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - DAS decay associated spectrum - PMSF phenylmethylsulfonyl fluoride     

Reconstitution of the DNA base excision—repair pathway

Background: The base excision–repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision–repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision–repair, we have attempted to reconstitute the pathway in vitro using purified enzymes.Results We report here the successful reconstitution of the base excision–repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5′ terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5′ nuclease function of DNA polymerase I.Conclusion Repair of uracil in DNA is achieved by a very short-patch excision–repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches.     

Rat liver foci study on coexposure with 50 Hz magnetic fields and known carcinogens

A study was performed to investigate possible interactions by magnetic fields (MF) with the processes of initiation and promotion of chemically induced preneoplastic lesions in rat liver. Male Sprague-Dawley rats were subjected to a 70% partial hepatectomy followed after 24 h by i.p. injection of diethylnitrosamine (DENA) as a tumour initiator. Starting one week after the DENA-treatment phenobarbital (PB) was given to promote growth of enzymatically altered foci of liver cells. MF was applied immediately after the partial hepatectomy and continued until sacrifice after 12 weeks of PB exposure. Homogenous horizontal AC magnetic fields with a frequency of 50 Hz and flux densities of 0.5 μT or 0.5 mT were used. The rats coexposed with MF and DENA plus PB did not gain weight as much as the rats exposed to the chemical agents only. The MF-exposure also resulted in a slight reduction in size and numbers of the focal lesions. The results suggest an interaction of MF with the processes of chemical carcinogenesis either as a result of stress or depending on effects on the proliferation of preneoplastic cells. © 1993 Wiley-Liss, Inc.     

Distribution of GABA-like immunoreactivity during post-metamorphic development and regeneration of the central nervous system in the ascidian Ciona intestinalis

During regeneration of the neural ganglion in Ciona intestinalis, the pattern of reappearance of some peptidergic cells is similar to the ontogenetic patterns exhibited by these cell types during normal post-metamorphic development. Using a specific antiserum to gamma-aminobutyric acid (GABA), we describe here the appearance of GABA-ergic cells in Ciona during both post-metamorphic development and regeneration of the neural ganglion following total ablation. Post-metamorphic animals were divided into the categories: 1, 3–5, 6–10, 11–15 and 23–27 mm in body length. Regeneration was monitored at 12, 15, 18, 21, 28 and 56 days post ablation. The first appearance of GABA-like immunoreactive cells during normal development were at the 3 to 5-mm stage where they were seen as discrete cells, without processes, evenly distributed in the cortical region throughout the ganglion. Fibres were first seen at the 6 to 10-mm stage. As development proceeded, GABA-like immunoreactive cells became more concentrated near the nerve root exits and along the dorsal rind of the ganglion. In regenerating ganglia, GABA was first detected at 18–21 days post ablation, in cells that lacked any obvious processes and that were distributed in all regions of the ganglion. At 28 days post ablation, processes could be detected in the neuropile, and after 56 days GABA cells were found predominantly in the same regions as in the normally developing adult ganglion. Although the overall pattern reflects that in a normal adult, a few differences were detectable. For example, rather more GABAergic cells were concentrated ventrally in the ganglion close to the neural gland.     

Laminin and Neuropeptide Y Are Increased by Synapsin Transfection in Cultured NG108-15 Neuroblastoma/Glioma Hybrid Cells

Abstract: We have investigated the presence and expression of laminin and neuropeptide Y (NPY) in several NG108-15 cell lines transfected with synapsin Ib, IIa, or IIb. The content of laminin, a basal membrane glycoprotein that promotes adhesion and induces neurite outgrowth and neuronal differentiation, was increased in all transfected cell lines examined. In cells that were chemically differentiated with prostaglandin E₁ plus 3-isobutyl-1-methylxanthine, laminin levels were increased even further. The content of NPY, suggested to be a neurotransmitter/neuromodulator in peripheral sympathetic neurons as well as in central neurons, was also increased in all transfected cell lines examined. Immunohistochemical analysis combined with confocal laser microscopy showed that NPY staining was granular and very often enriched in neuritic varicosities. The distribution and the staining pattern of NPY were consistent with storage of NPY in large dense-cored vesicles. The results indicate that, in differentiated neurons, the synapsins increase the levels of a neuropeptide transmitter stored in large dense-cored vesicles and of an extracellular matrix protein associated with neuronal maturation.     

Development of regulation mechanisms for SO42- influx in spring wheat roots

The development of SO₄^2- influx in roots and sulfur transport to shoots was followed in ³⁵S-tracer experiments for sulfur-deficient spring wheat (Triticum aestivum L. cv. Svenno) seedlings pretreated for various time periods (0–24 h) in nutrient solutions with SO₄^2-. Effects of the metabolic inhibitor 2,4-dinitrophenol (DNP) and the protein synthesis inhibitor cycloheximide (CH) on SO₄^2- influx were also evaluated. The SO₄^2- influx appears feedback-regulated by the internal sulfur level of the roots. Regulation may be achieved solely by a rapidly changed SO₄^2- carrier activity through an allosteric effect by the intracellular SO₄^2- concentration of the roots, followed first by induction of carrier synthesis and then by repression of carrier synthesis after transfer of the roots from SO₄^2--deficient nutrient solutions to solutions with SO₄^2-. A Hill plot of the partly sigmoidal relationship between SO₄^2- influx and intracellular sulfur concentration in the roots gave a Hill coefficient of -4.2, indicating negative cooperativity between a minimum number of four interacting allosteric binding sites for sulfur on each carrier entity. DNP-experiments showed that SO₄^2- influx was mainly metabolic, especially after short pretreatment in SO₄^2- at an external SO₄^2- concentration of 0.1 mM. Pretreatment with CH rapidly prevented new SO₄^2- carriers from being formed. Long CH pretreatment (24 h) and different SO₄^2- pretreatments reduced SO₄^2- influx below the non-metabolic level obtained by uptake experiments with DNP, indicating the existence of SO₄^2- carriers mediating passive SO₄^2- transport across the plasmalemma of the root cells. SO₄^2- influx was further decreased for the CH pretreated (24 h) plants by the presence of both CH and DNP in the experimental nutrient solution. This probably indicates the diffusive part of the non-metabolic SO₄^2- influx in the present experiments. Finally, it is suggested that there is a feedback signal between root and shoot, regulating sulfur transport upwards.     

Calbindin D-28k-immunoreactivity in rat muscle spindles during postnatal maturation and after denervation

Summary Calbindin D-28k-immunoreactivity has been demonstrated in some of the intrafusal muscle fibres and in the capsule of adult rat muscle spindles. In this study, the immunocytochemical localization of calbindin D-28k in the muscle spindles of triceps surae muscle was studied during postnatal maturation and after denervation. In young rats calbindin D-28k-immunoreactivity was seen in a few intrafusal fibres, first at the age of 4 days. At the 7th day, three calbindin D-28k-immunoreactive fibres and one unlabelled fibre were seen in most muscle spindles, as in adult rats. The spindle capsule and perineurial sheath of nerves were first seen to exhibit calbindin D-28k immunoreactivity at the age of 14 days, and thereafter the localization of calbinding D-28k-like immunoreactivity was similar to that in adult rats. After denervation, calbindin D-28k-immunoreactivity remained in intrafusal muscle fibres and the spindle capsule for a long period. After two months of denervation, calbindin D-28k immunoreactivity could still be seen in the spindle capsule, but the intrafusal fibres were not labelled.The innervation is known to have trophic effects on the intrafusal fibres. The present findings suggest that the expression of calbindin D-28k-immunoreactivity in maturating muscle spindles may be induced by the developing innervation. The decrease of calbindin D-28k-immunoreactivity in intrafusal fibres after denervation may be due to the loss of trophic factors released by the nerves.     

A rapid method for the analysis of N tau-methylhistidine in human urine

A novel, automated method is described for the determination of N^τ-methylhistidine in human urine. The method uses a modification (H. Nakamura and J. J. Pisano (1976) Arch. Biochem. Biophys.177, 334–335) of the reaction of fluorescamine with amines, which renders it specific for certain imidazoles. Interference due to histidine and histamine is selectively removed by prior reaction with aldehydes. The fluorescence yield for N^τ-methylhistamine is 280, 440, 50, and 1.7, respectively. The concentrations of N^τ-methylhistidine in human urine as determined by this technique correlate well (r = 0.99) with those determined by ion-exchange chromatography. Furthermore, the technique is rapid (6–7 samples/h net throughout), is reproducible (coefficient of variation 1.8%), requires no prior treatment of the sample, and is implemented with widely available equipment.     

Domain Organization,Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases

Cystathionine beta-synthase (CBS) is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS), fruit fly (dCBS) and yeast (yCBS) enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme’s catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet); however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme’s catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.