Divergence in ectomycorrhizal communities with foreign Douglas-fir populations and implications for assisted migration.

Assisted migration of forest trees has been widely proposed as a climate change adaptation strategy, but moving tree populations to match anticipated future climates may disrupt the geographically based, coevolved association suggested to exist between host trees and ectomycorrhizal fungal (EMF) communities. We explored this issue by examining the consistency of EMF communities among populations of 40 year-old Douglas-fir (Pseudotsuga menziesii var. menziesii) trees in a common-garden field trial using four provenances from contrasting coastal climates in southwestern British Columbia. Considerable variation in EMF community composition within test sites was found, ranging from 0.38 to 0.65 in the mean similarity index, and the divergence in EMF communities from local populations increased with site productivity. Clinal patterns in colonization success were detected for generalist and specialist EMF species on only the two productive test sites. Host population effects were limited to EMF species abundance rather than species loss, as richness per site averaged 15.0 among provenances and did not differ by transfer extent (up to 450 km), while Shannon's diversity index declined slightly. Large differences in colonization rates of specialist fungi, such as Tomentella stuposa and Clavulina cristata, raise the possibility that EMF communities maladapted to soil conditions contributed to the inferior growth of some host populations on productive sites. The results of the study suggest locally based specificity in host-fungal communities is likely a contributing factor in the outcome of provenance trials, and should be a consideration in analyzing seed-transfer effects and developing strategies for assisted migration.

Intermittent claudication due to ischaemia of the lumbosacral plexus.

The distinct clinical syndrome of exercise induced ischaemia of the lumbosacral plexus is not a widely known cause for intermittent claudication. Eight patients with the mentioned syndrome were investigated clinically, neurophysiologically, and with imaging techniques. The clinical examination showed a typical exercise induced sequence of symptoms: pain, paraesthesia, and sensory and motor deficits. The underlying vascular conditions were high grade stenoses or occlusions of the arteries supplying the lumbosacral plexus. Spinal stenosis could be excluded in all cases. Five patients received successful interventional radiological therapy. The syndrome can be diagnosed clinically and successful therapy is possible by interventional radiology.

Lovastatin induces mitotic abnormalities in various cell lines.

We examined the effects of lovastatin, a common anti-atherosclerotic drug and a blocker of the cell cycle, on the process of mitosis. It is known that lovastatin induces an arrest or a retardation of the cell cycle in many cell types not only at the G(1)phase, but also at the G(2)/M transition. After 24-48 h incubation of epithelial PtK(2), T24, HeLa cells and fibroblastic L929 cells in the presence of 1. 0-60.0 microm lovastatin, diverse mitotic perturbations have been observed. The most noteworthy phenomena recorded were prometaphase retardation and chromosome lagging during metaphase and anaphase. After the recovery in lovastatin-free media, the cells continued mitosis without any disturbances. Mevalonic acid prevented the effects of lovastatin. We conclude that the effects were specific for lovastatin-induced inhibition of mevalonic acid synthesis. Immunofluorescence studies with anticentromeric antibodies suggested that one of the possible causes of the lovastatin-induced mitotic disorder could be an interference with the development and function of the centromeres.

Levels of genetic diversity at different stages of the domestication cycle of interior spruce in British Columbia.

Concerns over the reductionist nature of the domestication of forest-tree species focus on the possibility of potential genetic erosion during this process. To address these concerns, genetic diversity assessments in a breeding zone the Province of British Columbia "interior" spruce (Picea glauca x engelmanni) program was conducted using allozyme markers. Genetic-variation comparisons were made between natural and production (seed orchard) populations as well as seed and seedling crops produced from the same breeding zone's seed orchard. The natural population sample consisted of a total of 360 trees representing three stands within each of three watersheds present in the Shuswap-Adams low-elevation zone of interior British Columbia. Small amounts of genetic differentiation were observed among the nine natural populations (4%) and this was attributable to extensive gene flow (N(m) = 7). Consequently, the sum of these nine populations was considered as a baseline for the genetic variation present in the breeding zone. The comparisons between the seed orchard and the breeding zone produced a similar percentage of polymorphic loci (%P = 64.7%) while the expected hetrozygosity (H(e)) (0.207 vs 0.210) and the average number of alleles per locus (2.7 vs 2.4) were slightly lower in the seed orchard. A total of seven natural populations' rare alleles (P < 0.007) were not present in the orchard population, while one allele was unique to the orchard. The %P increased to 70.6% in the seedlot, but dropped to the natural populations level (64.7%) in the plantation. The observed increase in %P was a result of pollen contamination in the orchard. It is suspected that the reduction in the plantation was caused by an unintentional selection in the nursery. Simulated roguing in the orchard did not drastically reduce H(e) even if up to 50% of the orchard's clones were rogued. However, roguing was associated with a reduction in the average number of alleles per locus (i.e., sampling effect).

An inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces arrest in G2-phase and metaphase in HeLa cells.

HeLa cells were treated with different concentrations of an inhibitor of the proteasome chymotrypsin-like activity, the peptidyl aldehyde N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). A detailed analysis, which included flow cytometry, cell counting and morphological assessment, was performed. PSI treatment induces a significant reduction of mitotic activity, accompanied by metaphase arrest of the mitotic cells. DNA flow cytometry shows an accumulation of the cells in G2+M phases of the cell cycle, which indicates the existence of a proteasome-mediated step in the G2-phase of the cell cycle. After removal of the inhibitor and supplementation with fresh medium, the cell cycle is resumed, but the mitotic cells show increased misalignment of chromosomes in the metaphase plate. PSI also induces HeLa cells to acquire a fibroblastoid phenotype.

A critical appraisal of synchronization methods applied to achieve maximal enrichment of HeLa cells in specific cell cycle phases.

Synchronization of mammalian cell cultures is a prerequisite for studies of molecular mechanisms of cell cycle control. Many researchers routinely use widely spread tumor cell lines like HeLa for these purposes, and a great variety of synchronization protocols has been described. Generally, they have been developed for monolayer cultures, usually with satisfactory results. However, we found that is not necessarily the case for cells cultivated in suspension. A critical appraisal of different standardized methods for selective enrichment of HeLa cells in suspension in all phases of the cell cycle has been undertaken. Our results reveal that only a few of the applied procedures can really yield high numbers of synchronized cells in G1, S, G2, and M phases, working with suspension cultures.

Restriction map and polymorphisms of nuclear ribosomal genes of Populus balsamifera.

Balsam poplar (Populus balsamifera) clones from five populations, which were collected along a transect from northern Wisconsin to the northern tree line, were evaluated for polymorphisms in nuclear ribosomal DNA. For this purpose, a restriction map was constructed using four six-cutter enzymes in single and double digests of genomic DNA. After electrophoretic separation on agarose gels and Southern transfer, blots were hybridized to non-radioactively labeled heterologous rDNA probes of soybean. Among populations, variation was detected in the length of the intergenic spacer between the tandem repeats of the coding regions and in the degree of methylation of one restriction enzyme recognition site. Based on a comparison of the derived restriction map of balsam poplar and other poplars, high homology was evident in the rDNA coding regions among species, whereas the intergenic spacer varied slightly in both length and number of restriction sites.

A novel nuclear inhibitor I-92 regulates DNA binding activity of octamer binding protein p92 during the cell cycle.

Nuclear DNA binding protein p92 is a sequence specific octamer binding protein with identical molecular weight as the ubiquitous octamer binding protein Oct-1. It binds to octamer related sequences from the enhancer of human papillomavirus type 18. The activity and intracellular distribution of p92 is regulated by extracellular signals. In serum starved Hela-fibroblast hybrid cells p92 is localized to the cytosol. Serum stimulation leads to nuclear import of p92. In fractions of asynchronously growing cells, which were separated according to cell cycle phases into G1, S, and G2 populations by centrifugal elutriation, p92 DNA binding is confined to S phase. In binding site blots however, p92 DNA binding activity is also present in G1 and G2. In G1 and G2 DNA binding activity of p92 is masked by a novel nuclear inhibitor I-92. The cyclic association of p92 with its inhibitor I-92 provides a new mechanism of regulating S phase dependent activity of a sequence specific DNA binding protein.

Flow cytometric analysis of Epstein-Barr virus (EBV) latent membrane protein expression in EBV-infected Raji cells.

In cultures of the latently Epstein-Barr virus (EBV)-infected Burkitt's lymphoma cell line Raji, the detectable amount of the EBV-encoded latent membrane protein (LMP) is transiently increased after addition of fresh nutrient medium containing foetal calf serum. In the current study, the relative amount of LMP and DNA in Raji cells was determined by biparametric flow cytometry analysis at different times after the addition of fresh medium with 10% foetal calf serum to a dense Raji culture. A transient increase in the proportion of LMP-positive cells was observed during the lag phase of the culture. Subsequently, a subpopulation of cells, which had been arrested in the G0 or G1 phase, simultaneously started to progress through the cell cycle. Neither the amount of LMP in the cells, nor the enhanced expression of LMP, was restricted to a certain phase of the cell cycle. Further analysis revealed that the number of LMP-positive cells proceeding simultaneously from the G1 to the S phase of the cell cycle is about the same as the total number of cells changing phases. These results suggest that LMP expression might be one step in the pathway leading to growth activation of resting cells in cultures of the immortalized Raji cell line.

Genetic evaluation of haploid clonal lines of a single donor plant of Populus maximowiczii.

Haploid clonal lines derived from anthers of a single donor plant of Populus maximowiczii were evaluated for several quantitative traits. In a nursery test, variances due to clonal lines ranged from 8% to 12% of the phenotypic variance in growth cessation and flushing date, respectively. No variance in relative shoot growth rate was associated with clonal lines. In a greenhouse study, gametoclonal variance in several leaf morphology traits ranged from 9% to 37% of the total variance. In relative wood density, variation due to haploid lines accounted for 25% of the total variance. In an isozyme analysis of 20 haploid and dihaploid plants, significant non-Mendelian segregation in isocitrate dehydrogenase was detected. The implications of these results for tree breeding are discussed.

Low density lipoprotein receptor-dependent prostaglandin synthesis in Swiss 3T3 cells stimulated by platelet-derived growth factor.

We studied the effects of human plasma lipoproteins on the synthesis of prostaglandin (PG) E2 in Swiss 3T3 mouse fibroblasts. Quiescent cells, maintained in medium deficient in both platelet-derived growth factor (PDGF) and lipoproteins, synthesized less than 8 ng of PGE2 per 10(6) cells per 22 hr, and this rate did not change in response to the addition of lipoproteins. In contrast, PDGF-stimulated cells, incubated in medium deficient in lipoproteins, synthesized 45-110 ng of PGE2 per 10(6) cells during the same period of time, and this rate increased 2- to 5-fold in the presence of added low density lipoproteins (LDL). This stimulatory effect of LDL seemed to depend on LDL receptor-mediated binding, uptake, and degradation of the lipoproteins because: both LDL and very low density lipoproteins were active, whereas high density lipoproteins were not; low concentrations of LDL were effective; the effect of native LDL was blocked by acetylation of the LDL; PDGF increased both the expression of LDL receptors and the cellular uptake of LDL; chloroquine blocked the effect of LDL but not that of exogenous arachidonic acid. These results provide evidence that the LDL pathway is critically linked to PG synthesis in PDGF-stimulated cells.

Detection and separation of the submetacentric marker chromosome of the WALKER (W-256) carcinoma using flow cytometry and sorting.

The chromosomes of WALKER (W-256) carcinoma cells have been separated into different DNA subclasses using DAPI for quantitative DNA staining and laser flow cytometry. The submetacentric marker chromosome could be isolated and its DNA content was determined to be 1.3 pg. One microgram marker DNA was obtained after separation of about 750 000 marker chromosomes by means of electronic flow sorting. The chromosomal composition of sorted fractions was analyzed by microscopy following banding of sorted chromosomes. The average morphological purity obtained was about 83%.

Cholesterol-induced growth stimulation, cell aggregation, and membrane properties of ascites tumor cells in culture.

Ascites tumor cells can be cultivated at a reduced serum concentration if cholesterol (2.50 mg per 100 ml of medium) is added to the culture medium. At serum concentrations of 3%, optimal growth properties are obtained; below 3%, cell cultures usually perish after a few days. Cells grown in the presence of added cholesterol have an elevated content of this molecule per cell as well as in the plasma membrane, and they also show a cholesterol concentration-dependent rate of proliferation. Precursors of the cholesterol-biosynthetic pathway like mevalonic acid, added in mM amounts, or squalene and lanosterol cannot be substituted for cholesterol itself. This is due to the observation that the biosynthetic pathway is blocked at the stage of lanosterol conversion to cholesterol. Cholesterol de novo synthesis from acetate is regulated by the cholesterol content of the cells, which also affects the production of ubiquinone and dolichol. Growth factors such as insulin, prostaglandin F2 alpha, and transferrin added to the medium do not mimic the cholesterol-induced effect. Distribution of DNA during cell cycle and the cell density-dependent reduction in macromolecule synthesis is very similar to the control cells. In contrast, cells without added cholesterol show reduced growth properties accompanied by the accumulation of cells in the mitotic and G2 phase. The cholesterol/phospholipid ratio of the plasma membranes of cholesterol-rich cells is about 15% lower than of the control cells and 40% higher compared to the cholesterol-poor cells, which, however, does not significantly alter the membrane fluidity between the cholesterol-rich and -poor cells as revealed by fluorescence polarization measurements. The most dramatic behavior of the cholesterol-rich cells is their tendency to form aggregates, which is demonstrated either by concanavalin A-induced agglutination or by cell density-dependent aggregation shown by interference microscopy in vivo.

Cytostatic and cytotoxic response of Ehrlich ascites tumor cells in vivo on chronic treatment with cytarabine, bleomycin, and peplomycin.

The cytokinetic response of Ehrlich ascites tumor (EAT) cells in vivo upon chronic treatment at low dosage levels with cytarabine (1-beta-D-arabinofuranosylcytosine, ara-c) bleomycin (BLM) and peplomycin (PEP) was estimated. Bivariate DNA histograms allow the simultaneous evaluation of the cell cycle status of living and killed cells. It could be confirmed that ara-C is cytostatic on cells in S phase. Pronounced cytotoxicity was observed in G1 and G2+M phase. BLM and PEP showed no (or neglectable ) accumulation of vital cells in any cycle phase. Both drugs, however, are cytotoxic on cells, regardless their position within the cell cycle. A successive application of ara-C and BLM (or PEP) in a cell kinetics-directed therapy schedule may be taken into account.

Chromosomes for molecular hybridization. Assignment of repetitive and single copy genes using a rapid filter-fixation method.

Specific recombinant DNA sequences (5S rRNA, B1, albumin) were assigned to flow sorted chromosomes of the Chinese hamster cell line CHV79. For this purpose, a rapid protocol was developed using filterbound chromosomal DNA and probing with various nucleic acids, that allows sequence identification in chromosomes. A flow histogram and a flow karyogram of the CHV79 cell line were established by flow analysis in order to calculate the amount of DNA per CHV79 cell and their chromosomes. Subsequently, metaphase chromosomes or chromosomal groups were fractionated by electronic sorting and a defined number of chromosomes was directly bound to nitrocellulose filters for sequence homology analysis by a dot blot hybridization procedure. This procedure not only allows the assigning of specific DNA sequences to particular chromosomes, it is also applicable to studies of changes in karyotypes, for example translocations of given sequences.

A reliable preparation of mon-dispersed chromosome suspensions for flow cytometry.

A reproducible, simple and rapid protocol was developed to prepare mono-dispersed chromosome suspensions for flow cytometric analysis. The procedure basically employes: 1. twice rinsing of monolayer cultures to eliminate dead cells and cellular debris, 2. mild fixation of mitotic cells with 1% acetic acid, and 3. ultra sonic treatment to release single metaphase chromosomes. The procedure takes less than 30 min. Isolated chromosomes are storable over months at 4 degrees C. Despite mild fixation many biochemical studies and experiments applied on such fixed chromosomes purified and enriched using electronic sorting are feasible: 1. gene and restriction mapping, 2. cloning of specific gene sequences, and 3. gene frequency analysis.

A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis.

The amplified, extrachromosomal nucleoli of Xenopus oocytes contain a meshwork of approximately 4-nm-thick filaments, which are densely coiled into higher-order fibrils of diameter 30-40 nm and are resistant to treatment with high- and low-salt concentrations, nucleases (DNase I, pancreatic RNase, micrococcal nuclease), sulfhydryl agents, and various nonionic detergents. This filamentous "skeleton" has been prepared from manually isolated nuclear contents and nucleoli as well as from nucleoli isolated by fluorescence-activated particle sorting. The nucleolar skeletons are observed in light and electron microscopy and are characterized by ravels of filaments that are especially densely packed in the nucleolar cortex. DNA as well as RNA are not constituents of this structure, and precursors to ribosomal RNAs are completely removed from the extraction-resistant filaments by treatment with high-salt buffer or RNase. Fractions of isolated nucleolar skeletons show specific enrichment of an acidic major protein of 145,000 mol wt and an apparent pI value of approximately 6.15, accompanied in some preparations by various amounts of minor proteins. The demonstration of this skeletal structure in "free" extrachromosomal nucleoli excludes the problem of contaminations by nonnucleolar material such as perinucleolar heterochromatin normally encountered in studies of nucleoli from somatic cells. It is suggested that this insoluble protein filament complex forms a skeleton specific to the nucleolus proper that is different from other extraction-resistant components of the nucleus such as matrix and lamina and is involved in the spatial organization of the nucleolar chromatin and its transcriptional products.