Kälteresistenz der Fichte

Fichtenchloroplasten durchlaufen während eines Jahres saisonbedingte charakteristische Struktur- und Funktionswechsel: Frühjahrschloroplasten, die in den alten Nadeln kurz vor und während des Knospenaustriebes gefunden werden, sind sehr groß und so sehr mit Stärke erfüllt, daß man sie als Amyloplasten bezeichnen kann. Beim Aufbau der neuen Nadelgeneration wird diese Stärke verbraucht, und es entwickeln sich aus den Amyloplasten wieder die photosynthetisch aktiven Sommerpiastiden mit einem gut entwickelten Membransystem. Während der Frosthärtung und insbesondere während der Frostperiode treten die Chloroplasten in ihrer Winterform auf: amöboide, an einer Stelle der Zelle konzentrierte, stark aufgequollene Plastiden mit einem aufgelockerten und zum Teil reduzierten Thylakoidsystem. Der Strukturwandel der Chloroplasten wird — wie die Frostresistenz selbst — durch exogene Faktoren (Tageslänge, Temperatur) ausgelöst und kann auch durch artifiziell veränderte Umgebung zu unnatürlichen Zeitpunkten ausgelöst werden. Begleitet wird der Strukturwandel von einer Veränderung der CO₂-Fixierungsrate der Fichtennadeln, die ihrerseits auf Veränderungen der photochemischen Aktivität der Chloroplasten zurückzuführen ist. In Frostexperimenten konnte gezeigt werden, daß die Frosthärtung Reaktionen auf zwei verschiedenen Ebenen auslöst: 1. Produktion von kolligativ wirksamen Membranschutzstoffen sowie 2. einer Veränderung der chemischen Zusammensetzung der Chloroplastenmembranen; zumindest letztere führt offensichtlich zu einer Verringerung der Photosyntheseleistung. Frostschädigung der Chloroplasten tritt bei nicht entsprechend stark gehärteten Nadeln auf, jedoch nicht durch direkten Einfluß der tiefen Temperatur auf das Thylakoidsystem als vielmehr durch Freisetzung membranschädigender Substanzen durch Permeabilitätsverlust plasmatischer Membranen. Wir danken Herrn Prof. Dr. O. Kandler für anregende Diskussionen und für die kritische Durchsicht des Manuskriptes. Der Deutschen Forschungsgemeinschaft wird für die finanzielle Unterstützung dieser Arbeit gedankt.     

Subunit architecture of multimeric complexes isolated directly from cells

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.     

Barn owls (<Emphasis Type="Italic">Tyto alba</Emphasis>) in western North America: phylogeographic structure,connectivity, and genetic diversity

The barn owl (Tyto alba) is a non-migratory species widely distributed across much of North America in areas with extensive old-field and grassland habitat and without extensive winter snow cover. We investigated the genetic diversity and phylogeographic patterns of barn owl populations in western North America, ranging from British Columbia (BC) to southern California, and one eastern population from Pennsylvania. We also determined the genetic distinctiveness of a population off the coast of southern California, Santa Barbara Island, as management plans to control the local owl population are being considered to decrease predation rate on the now threatened Scripps’s Murrelet (Synthliboramphus scrippsi). Using 8 polymorphic microsatellite markers (N = 126) and ND2 mitochondrial sequences (N = 37), we found little to no genetic structure among all sampled regions, with the exception of Santa Barbara Island. The BC mainland population, despite its northwestern geographically peripheral location and ongoing habitat degradation, is not genetically depauperate. However, individuals from Vancouver Island, likewise a peripheral population in BC, exhibited the lowest genetic diversity of all sampled locations. The low global F_ST value (0.028) estimated from our study suggests that old-field agricultural habitats are well connected in North America. Since the BC population has declined by about 50 % within the last three decades, it is vital to focus on preserving the remaining barn owl habitats in BC to allow successful establishment from neighbouring populations. Additionally, our microsatellite data revealed that the population on Santa Barbara Island showed genetic divergence from its continental counterpart. Mitochondrial data, however, demonstrated that this island population is not a monophyletic lineage containing unique haplotypes, and hence cannot be designated as an Evolutionarily Significant Unit.     

Isolation and Preliminary Characterization of Bacteriophages for Bdellovibrio bacteriovorus

Ten bacteriophages that attack and lyse saprophytic strains of Bdellovibrio bacteriovorus were isolated. Morphological, serological, and host-range studies revealed that there were four different bdellovibrio phages present among the isolates. One of the phages lysed a strain of B. bacteriovorus that requires the presence of a suitable bacterial host for growth. The phage attached to the bdellovibrio cells in the absence of the bacterial host cells; lysis occurred only in the presence of host cells. The 19 saprophytic bdellovibrio strains employed in the phage host-range studies were grouped on the basis of their susceptibility to phage lysis.     

Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and ³¹P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. ³¹P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.     

DQ 65-79, a peptide derived from HLA class II, mimics p21 to block T cell proliferation

DQ 65-79, a peptide derived from residues 65-79 of the alpha-chain HLA class II molecule DQA03011, blocks T cell proliferation and induces T cell apoptosis. Using a yeast two-hybrid assay, we previously identified proliferating cell nuclear Ag (PCNA) as an intracellular ligand for DQ 65-79. In this study, we show that three regions of PCNA, residues 81-100, 121-140, and 241-261, interact with DQ 65-79. Residues 241-261 of PCNA also interact with the C terminus (residues 139-160) of the cell cycle regulator, p21, suggesting that DQ 65-79 and p21 might function similarly. We show here that DQ 65-79 competitively inhibits binding of p21 to PCNA and that both DQ 65-79 and p21 139-160 induce T cell apoptosis, suggesting that DQ 65-79 and p21 act similarly to inhibit cell growth.     

Phenotypic characterization of sym 21, a gene conditioning shoot-controlled inhibition of nodulation in Pisum sativum cv. Sparkle

E132 ( sym 21) is a stable pleiotropic mutant of Pisum sativum cv. Sparkle obtained by mutagenesis with ethyl methane sulfonic acid. The line forms few nodules and short, highly branched roots. Microscopy studies revealed that infection by rhizobia is normal, and low nodulation is mainly due to a low rate of emergence of the nodule meristems. E132 shoots depressed nodulation on Sparkle stocks, whereas in reciprocal grafts more nodules formed on E132 stocks than on control roots or self-grafted Sparkle plants. Nodule number on the mutant was slightly increased by exogenous ethylene inhibitors, which, however, did not alter the root phenotype.     

Influence of climate change and postdelisting management on long‐term population viability of the conservation‐reliant Kirtland's Warbler

Rapid global climate change is resulting in novel abiotic and biotic conditions and interactions. Identifying management strategies that maximize probability of long‐term persistence requires an understanding of the vulnerability of species to environmental changes. We sought to quantify the vulnerability of Kirtland's Warbler (Setophaga kirtlandii), a rare Neotropical migratory songbird that breeds almost exclusively in the Lower Peninsula of Michigan and winters in the Bahamian Archipelago, to projected environmental changes on the breeding and wintering grounds. We developed a population‐level simulation model that incorporates the influence of annual environmental conditions on the breeding and wintering grounds, and parameterized the model using empirical relationships. We simulated independent and additive effects of reduced breeding grounds habitat quantity and quality, and wintering grounds habitat quality, on population viability. Our results indicated the Kirtland's Warbler population is stable under current environmental and management conditions. Reduced breeding grounds habitat quantity resulted in reductions of the stable population size, but did not cause extinction under the scenarios we examined. In contrast, projected large reductions in wintering grounds precipitation caused the population to decline, with risk of extinction magnified when breeding habitat quantity or quality also decreased. Our study indicates that probability of long‐term persistence for Kirtland's Warbler will depend on climate change impacts to wintering grounds habitat quality and contributes to the growing literature documenting the importance of considering the full annual cycle for understanding population dynamics of migratory species.     

Identification of 57 conventional RFLP and 6 VNTR systems with 32 DNA clones on chromosome 11p15

Fifty-four clones containing human inserts were selected from a cosmid library constructed from a somatic cell hybrid containing chromosome 11p15.3-p15.5 as its only human complement. In 32 of these clones, 63 polymorphic systems were identified with a panel of restriction enzymes: 57 conventional RFLP systems and 6 highly polymorphic VNTR systems. Although we examined the cosmid with only seven enzymes, 18 clones (including 6 VNTRs) were polymorphic with three or more enzymes. The results suggested that DNA sequences on the peritelomeric region of chromosome 11p tend to be highly variable. Because these markers are highly informative, they will be excellent resources for investigations of hereditary diseases and tumor suppressor genes in this region of chromosome 11.