Ancient DNA from Bronze Age bones of European rabbit (Oryctolagus cuniculus)

The European rabbit (Oryctolagus cuniculus) is now widely distributed throughout the world as a result of transportation by man. The original populations, however, were confined to southern France and Spain. In order to investigate the role of human intervention in determining the genetic diversity of rabbit populations, we are studying the origin of rabbits introduced onto a small Mediterranean island (Zembra) near Tunis over 1400 years ago, by examining ancient DNA extracted from rabbit bones found both on Zembra and on the European mainland. Ancient DNA was successfully extracted from rabbit bones found at two archaeological sites dated to at least the Early Bronze Age (more than 3500 years ago) in south-central France, and compared to that found in modern mainland and island populations using a small variable region of the cytochromeb gene. The results confirm that the Zembra Island population is descended from that present over 1400 years ago. The technical aspects of DNA extraction from bones and the implications of this type of research for determining the origin of introduced rabbit populations are discussed.     

An analysis of allozyme,mitochondrial DNA and morphological variation in mussel (Mytilus galloprovincialis) populations from Greece

Three populations ofM. galloprovincialis from northern Greece were investigated using isozyme analysis, discriminant analysis of morphological characteristics and analysis of restriction fragments of mtDNA. For all three types of analysis significant intra- and interpopulation differentiation was found. This differentiation is very noticeable at the mtDNA genotype frequencies. Furthermore, the restriction patterns of mtDNA were different from those reported for Atlantic populations of this species.     

Disruption of mitochondrial function as the basis of the trypanocidal effect of trifluoperazine onTrypanosoma cruzi

Summary The tricyclic anti-calmodulin drug trifluoperazine (TFP) inhibited growth and motility of epimastigotes ofTrypanosoma cruzi, at concentrations lower than 100 M, and motility and infectivity of the bloodstream trypomastigote form at 200 M. Electron microscopy of TFP-treated epimastigotes showed that the major effect was at the mitochondrial level, with gross swelling and disorganization. The oligomycin-sensitive, mitochondrial ATPase was completely inhibited by 20 M TFP, and the same drug concentration caused a 60% decrease in intracellular ATP content. The results suggest that the trypanocidal effect of TFP may be related more to mitochondrial damage than to the well-known anticalmodulin effect of the drug.     

Random chromosome breakage by colchicine inViscum fischeri (Loranthaceae)

Summary When the shoot-tips ofViscum fischeri Engl. were treated with various concentrations of colchicine for different lengths of time, it was found that in this plant chromosome breakage was not localized to centromeric region as reported in other plants. InV. fischeri chromosome breakage occurred at random (simulating X-ray-induced fragmentation). The percentage of breakage increased linearly with respect to time at concentrations 0.1, 0.2, 0.3% but parabolically at 0.5%.Acknowledgment. The authors are grateful to Dr M. A. Hannan of NRC, Ottawa, Canada, for his helpful criticism and suggestions, and to Professor S. K. Imbamba, Chairman, Dept. of Botany, University of Nairobi for laboratory facilities.     

Mitochondrial distribution and inheritance

Mechanisms mediating the inheritance of mitochondria are poorly understood, but recent studies with the yeastsSaccharomyces cerevisiae andSchizosaccharomyces pombe have begun to identify components that facilitate this essential process. These components have been identified through the analysis of conditional yeast mutants that display aberrant mitochondrial distribution at restrictive conditions. The analysis of these mutants has uncovered several novel proteins that are localized either to cytoskeletal structures or to the mitochondria themselves. Many mitochondrial inheritance mutants also show altered mitochondrial morphology and defects in maintenance of the mitochondrial genome. Although some inheritance components and mechanisms appear to function specifically in certain types of cells, other conserved proteins are likely to mediate mitochondrial behavior in all eukaryotic cells.     

Reproductive competition and colony fragmentation in the guest-ant,Formicoxenus provancheri

Aggressive reproductive conflicts and dominance interactions among queens are involved in establishing functional monogyny in the ant,Formicoxenus provancheri. Competition among potential reproductives may lead to the founding of new societies by budding or colony fragmentation.     

Radial skeletal dissolution to promote vegetative reproduction in a solitary coralDiaseris distorta

The solitary, free-living coralDiaseris distorta (Michelin) (Fungiidae, Scleractinia) reproduces asexually by fragmentation along radially oriented slits. Localized skeletal dissolution, which can be recognized as white, opaque and chalky lines along the thecal wall between segments, ultimately results in autotomy. We suggest that the skeletal dissolution which dissolves the weakest part of the corallum for easy breakage, is a species-specific character.     

Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence

Phage display was used to identify new components of the mammalian mitochondrial receptor complex using Tom20 as a binding partner. Two peptides were identified. One had partial identity (SMLTVMA) with a bacterial signal peptide from Toho-1, a periplasmic protein. The other had partial identity with a mitochondrial inner membrane glutamate carrier. The bacterial signal peptide could carry a protein into mitochondria both in vivo and in vitro. The first six residues of the sequence, SMLTVM, were necessary for import but the two adjacent arginine residues in the 30-amino-acid leader were not critical for import. The signal peptides of Escherichia coli β-lactamase and Bacillsus subtilis lipase could not carry proteins into mitochondria. Presumably, the Toho-1 leader can adopt a structure compatible for recognition by the import apparatus.Received 29 April 2005; received after revision 8 June 2005; accepted 17 June 2005     

Partial purification and characterization of acid phosphatase from sporulated oocysts ofEimeria tenella

Summary Acid phosphatase ofEimeria tenella oocysts (Peak II) was purified 77-fold with a recovery of 26% using protamine sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. This enzyme occurs in multiple forms as indicated by two peaks which can be separated by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The partially purified enzyme has optimal activity at pH 4.5. With p-nitrophenyl phosphate the Km and V_max values for (Peak II) were 25 mM and 1.57 mol/min/mg protein, respectively. The enzyme (Peak II) ist strongly inhibited by Hg⁺⁺, Cu⁺⁺, iodoacetamide, fluoride and molybdate. Tartrate and other divalent metal ions have no effect on enzyme activity. The partially purified Peak II phosphatase is not a glycoprotein as it is not absorbed on concanavalin-A Sepharose and its treatment with bacterial neuraminidase does not alter its elution profile through DEAE cellulose.     

DNA fragmentation in leukocytes following repeated low dose sarin exposure in guinea pigs

The objective of this study was to determine levels of DNA fragmentation in blood leukocytes and parietal cortex from guinea pigs following repeated lowlevel exposure to the chemical warfare nerve agent (CWNA) sarin. Guinea pigs were injected (s.c.) once a day for 10 days with saline, or 0.1, 0.2, or 0.4 LD₅₀ (50% mean lethal dose) sarin dissolved in sterile physiological saline. Blood and parietal cortex was collected after injection at 0, 3, and 17 days recovery and evaluated for DNA fragmentation using single-cell gel electrophoresis (Comet assay). Cells were imaged using comet analysis software and three parameters of DNA fragmentation measured: tail length, percent DNA in the tail, and tail moment arm. Repeated low-dose exposure to sarin produced a dose-dependent response in leukocytes at 0 and 3 days post-exposure. There was a significant increase in all measures of DNA fragmentation at 0.2 and 0.4 LD₅₀, but not at 0.1 LD₅₀. There was no significant increase in DNA fragmentation in any of the groups at 17 days post-exposure. Sarin did not produce a systematic dose-dependent response in parietal cortex at any of the time points. However, significant increases in DNA fragmentation at 0.1 and 0.4 LD₅₀ were observed at 0 and 3 days post-exposure. All measures of DNA fragmentation in both leukocytes and neurons returned to control levels by 17 days post-exposure, indicating a small and non-persistent increase in DNA fragmentation following repeated low-level exposure to sarin. Received 23 July 2007; received after revision 23 August 2007; accepted 3 September 2007 Research was conducted in compliance with the Animal Welfare Act, and other Federal statutes and regulation relating to animals and experiments involving animals and adheres to the principles stated in the Guide for the Care and Use of Laboratory Animals, NIH publication 85-23. The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense (para 4-3), AR 360–365.     

Competitiveness and communication for effective inoculation byRhizobium,Bradyrhizobium and vesicular-arbuscular mycorrhiza fungi

After a short summary on the ecology and rhizosphere biology of symbiotic bacteria and vesicular-arbuscular (VA) mycorrhiza fungi and their application as microbial inocula, results on competitiveness and communication are summarized. Stress factors such as high temperature, low soil pH, aluminium concentrations and phytoalexins produced by the host plants were studied withRhizobium leguminosarum bv.phaseoli andRhizobium tropici onPhaseolus beans. Quantitative data for competitiveness were obtained by usinggus ⁺ (glucoronidase) labelled strains, which produce blue-coloured nodules. ForPhaseolus-nodulating rhizobia, a group specific DNA probe was also developed, which did not hybridize with more than 20 other common soil and rhizosphere bacteria. Results from several laboratories contributing to knowledge of signal exchange and communication in theRhizobium/Bradyrhizobium legume system are summarized in a new scheme, including also defense reactions at the early stages of legume nodule initiation. Stimulating effects of flavonoids on germination and growth of VA mycorrhiza fungi were also found. A constitutive antifungal compound in pea roots, -isoxazolinonyl-alanine, was characterized.     

Mitochondrial DNA evidence for the 19th century introduction of African honey bees into the United States

Since the introduction of an African subspecies into Brazil in the mid-1950's¹, descendent Africanized honey bees (Apis mellifera L.) have spread throughout the Neotropics and into temperate North America. Restriction enzyme analysis of 422 feral honey bee colonies collected from non-Africanized areas in the southern United States revealed that over 21% of them had mitochondrial DNA (mtDNA) derived from a European race established in North America by the 17th century, 77% of them had mtDNA common in honey bees maintained by beekeepers and about 1% exhibited African mtDNA. Further analysis revealed that the African mtDNA was derived from a north African subspecies imported to the US in the 19th century.     

Structural diversity of trypsin from different mosquito species feeding on vertebrate blood

Summary Mosquito trypsin was purified using a combination of ion exchange and affinity chromatography with the ligand soybean trypsin inhibitor. ThreeAedes and threeAnopheles species were tested, all of which are specialized in the digestion of vertebrate blood. Amino-terminal sequences of HPLC-purified trypsins fromAedes aegypti andAnopheles quadrimaculatus revealed homologies of 30–40% with vertebrate and other invertebrate proteases previously identified as serine-proteases. The purified mosquito trypsins have molecular masses between 25 kDa and 36 kDa, as determined by denaturing polyacrylamide electrophoresis, and are heterogeneous in size and number in the various species. The number of SDS-bands varies between 3 and 6 inAedes and between 1 and 3 inAnopheles. The specific activities, determined with the substrate TAME, range from 240 U/mg inAedes aegypti to 1065 U/mg inAnopheles quadrimaculatus. All mosquito trypsins tested have acidic isoelectric points between pH 3.5 and pH 5.4. No alkaline proteases were detected. Polyclonal antisera againstAedes aegypti andAnopheles albimanus trypsin do not cross-react with bovine trypsin. Cross-reactivity of the two sera with trypsin from six mosquito species suggests the presence of at least 2 enzyme families.     

Studies on the fungal phytotoxin victorin: Structures of three novel amino acids from the acid hydrolyzate

Summary The host-selective phytotoxin victorin, produced by the fungusCochliobolus victoriae, was found to be at least partially peptidic in nature, and did not contain victoxinine. The exact mass of the M-H ion was measured by FABMS as 795.1877. Derivatives of three major acid hydrolysis products were isolated. The structures of the corresponding amino acids were assigned as 2S,3R-3-hydroxyleucine, 5,5-dichloroleucine, and 3-hydroxylysine. A into victorin by the fungus in vivo.     

Development of DNA probes for cytotoxin and enterotoxin genes in enteric bacteria

Summary DNA probes to identify the genes encoding toxins in enteric bacteria have been developed. Use of these probes reduces the number of animals required for toxicity testing, as suspect bacteria can be directly tested for the presence of toxin. We have augmented the gene probes available by developing probes against theEscherichia coli enterotoxin LTII and shiga toxin fromShigella dysenteriae 1.The LTII gene fromE. coli 357900 was identified and characterised and a suitable internal probe was obtained. The LTII gene was found not to be common among enterobacteriae from various geographical locations. Isolates predominately of animal origin from Nigeria and Thailand hybridized with the probe.The shiga toxin gene was isolated fromS. dysenteriae 1 by a combination of in vivo and in vitro methods. An internal probe was identified and used against different serogroups ofShigella andE. coli isolated. The probe was found to hybridize withS. dysenteriae 1 isolates and also someS. flexneri andS. sonnei strains. Representatives were tested for toxin production and found to produce toxin at low levels.     

Osmotic adaptation of moderately halophilic methanogenic Archaeobacteria,and detection of cytosolicN,N-dimethylglycine

Methanohalophilus mahii SLP andMethanohalophilus halophilus Z-7982, two closely-related, moderately halophilic, methylotrophic methanogens, were tested for their adaptation to saline conditions. They grew in a wider range of salinities than previously reported, in a defined medium with as little as 0.1 M NaCl, and with a high as 4.0 M NaCl forM. halophilus and 4.5 M NaCl forM. mahii. Fastest growth occurred with 1.5 M NaCl forM. mahii and 1.0 M NaCl forM. halophilus. M. mahii also grew in media in which NaCl was replaced by sucrose or KCl as osmolytes up to the osmolal equivalent of 2 and 2.5 M NaCl (these media contained other sodium salts totaling about 0.1 M Na⁺). In media with either sucrose of KCl replacing NaCl,M. mahii grew fastest at osmolalities approximately equiosmolal to 1 M NaCl.M. mahii not only grew well at a wide range of osmosities, it also tolerated rapid shifts in osmolality. Cells subjected to a rapid 10-fold hypertonic shift resumed growth without a prolonged lag. When cells were subjected to a rapid 10-fold hypotonic shift, 90% of cells lysed, but the remaineder continued to swell with little further lysis during the next 45 min. Surviving cells resumed growth.Methanohalophilus strains grown in defined medium had low cytosolic Na⁺ concentrations; K⁺ concentrations were as high as 0.35 M. Organic osmotica in the cytosol include glycine betaine and larger amounts of N,N-dimethylglycine.     

Histidine-induced injury to cultured liver cells,effects of histidine derivatives and of iron chelators

The amino acid histidine is an excellent buffer and is therefore included in several organ preservation solutions used in transplantation medicine. However, when used at concentrations as in these solutions, histidine has a marked injurious potential. Therefore, we here assessed the mechanism of histidine-induced cell injury and searched for ways to use the buffering power of histidine but avoid histidine toxicity. When cultured hepatocytes were incubated in HTK solution or in modified Krebs-Henseleit buffer containing 198 mM L-histidine at 37°C, most cells lost viability within 3 h (LDH release 86 ± 7% and 89 ± 5%, respectively). This injury was accompanied by marked lipid peroxidation, and was strongly inhibited by hypoxia, by the antioxidants trolox, butylated hydroxytoluene and N-acetylcysteine and by the membrane-permeable iron chelators 2,2′-dipyridyl, 1,10-phenanthroline, LK 614, LK 616 and deferoxamine. Thus, histidine-induced cell injury appears to be mediated by an iron-dependent formation of reactive oxygen species. D-Histidine, imidazol and L-histidine methyl ester also elicited marked injury, while the N-substituted derivatives Nα-acetyl-L-histidine and tert-butyl-oxycarbonylhistidine and histidine-containing dipeptides showed almost no toxicity. Histidine toxicity, its iron dependence and the superiority of Nα-acetyl-L-histidine were also evident during/after cold (4°C) incubations. Therefore, we suggest the addition of iron chelators to histidine-containing solutions, and/or replacing histidine with Nα-acetyl-L-histidine in organ preservation solutions. Received 23 October 2006; accepted 21 November 2006     

What leads from <Emphasis Type="Italic">dead-end?</Emphasis>

The 129 mouse strain develops congenital testicular germ cell tumors (TGCTs) at a low frequency. TGCTs in mice resemble the testicular tumors (teratomas) that occur in human infants. The genes that cause these tumors in 129 have not been identified. The defect at the Ter locus increases TGCT incidence such that 94% of 129-Ter/Ter males develop TGCTs. The primary effect of the Ter mutation is progressive loss of primordial germ cells (PGCs) during embryonic development. This results in sterility in adult Ter/Ter mice on all mouse strain backgrounds. However, on the 129 background, Ter causes tumor development in addition to sterility. Therefore, Ter acts as a modifier of 129-derived TGCT susceptibility genes. Ter was identified to be a mutation that inactivates the Dead-end1 (Dnd1) gene. In this perspective, I discuss the possible areas of future investigations to elucidate the mechanism of TGCT development due to Dnd1 inactivation. Received 29 September 2006; received after revision 29 January 2007; accepted 19 February 2007     

Criteria for permissible browse impact on sycamore maple (Acer pseudoplatanus) in mountain forests

Summary A naturally regenerated young-growth in the Buchser Hochwald area in the Canton of St. Gallen, Switzerland, was used to determine the silviculturally permissible browsing limit for sycamore maple (Acer pseudoplatanus). The investigated area is situated at an altitude ranging from 1280 to 1310 m a.s.l. on anAbieti-Fagetum typicum site. Browsing was done by red deer (Cervus elaphus) and roe deer (Capreolus capreolus). A total of 57 sycamore maples, 1.30 m high, were examined. The actual browse impact on these plants was assessed by determining the frequency of browsing marks on the stem axis. To this end, the plants were cut into sections of 5 cm each and then split radially. On this basis it was possible to calculate permissible shares of browsed plants for four different size categories between 0.10 and 1.30 m. The permissible share of plants with two or more visible browsing marks on the stem axis amounted to 37.8% as an average value for the total risk period. This corresponds to a quota of 26.9% browsed terminal shoots per annum.     

EGF-induced programmed cell death of human mammary carcinoma MDA-MB-468 cells is preceded by activation of AP-1

MDA-MB-468 is a human mammary adenocarcinoma cell line that overexpresses the epidermal growth factor (EGF) receptor and undergoes programmed cell death (apoptosis) in response to EGF treatment. Programmed cell death was shown to be greatly enhanced when cells were growth-arrested prior to EGF treatment. Apoptosis was characterized by an initial rounding up and detachment of the cells from their substrate starting about 12 h after EGF treatment, followed by chromatin condensation, nuclear fragmentation and oligonucleosomal fragmentation of the DNA at about 24 to 48 h. Cell death was dependent on de novo protein synthesis. We found a rapid induction of c-fos, c-jun and junB at the mRNA level after about 30 min of EGF treatment and a more delayed upregulation of fosB and fra-1. The junD gene was expressed in the absence of EGF, and it was moderately induced within 30 min of growth factor addition. The increase of the different fos and jun mRNAs were paralleled by an increase of activator protein-1 (AP-1) DNA binding activity. A characterization of the AP-1 complex revealed similar levels of several Fos and Jun proteins. Based on the kinetics of AP-1 accumulation and cell death, it seems likely that AP-1 contributes to the apoptotic cell death of EGF receptor-overexpressing MDA-MB-468 cells. Received 21 July 1997; received after revision 6 November 1997; accepted 6 November 1997