Effects of trehalose supplementation on cell viability and oxidative stress variables in frozen-thawed bovine calf testicular tissue

Trehalose is widely used for cryopreservation of various cells and tissues. Until now, the effect of trehalose supplementation on cell viability and antioxidant enzyme activity in frozen-thawed bovine calf testicular tissue remains unexplored. The objective of the present study was to compare the effect of varying doses of trehalose in cryomedia on cell viability and key antioxidant enzymes activities in frozen-thawed bovine calf testicular tissue. Bovine calf testicular tissue samples were collected and cryopreserved in the cryomedias containing varying doses (0, 5, 10, 15, 20 and 25%; v/v) of trehalose, respectively. Cell viability, total antioxidant capacity (T-AOC) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione (GSH) content and malondialdehyde (MDA) content were measured and analyzed. The results showed that cell viability, T-AOC activity, SOD activity, CAT activity and GSH content of frozen-thawed bovine calf testicular tissue was decreased compared with that of fresh group (P < 0.05). MDA content in frozen-thawed bovine calf testicular tissue was significantly increased compared with that of fresh group (P < 0.05). The cryomedia added 15% trehalose exhibited the greatest percentage of cell viability and antioxidant enzyme activity (SOD and CAT) among frozen-thawed groups (P < 0.05). Meanwhile, GSH content was the lowest among frozen-thawed groups (P < 0.05). However, there were no significance differences in MDA content among the groups added 10, 15 and 20% trehalose (P > 0.05). In conclusion, the cryomedia added 15% trehalose reduced the oxidative stress and improved the cryoprotective effect of bovine calf testicular tissue. Further studies are required to obtain more concrete results on the determination of antioxidant capacity of trehalose in frozen-thawed bovine calf testicular tissue.     

A kinetic model for bacterial transfection: the lambda DNA system.

A kinetic model describing the binding and uptake of free lambda phage DNA by the bacterium Escherichia coli is presented. The model is based on the assumption that adsorbed ‘helper’ phage particles serve as functional sites to which the lambda DNA specifically binds. When applied to experimental data, the model describes the reaction between cells and DNA as a rapid binding of DNA to helper phage attachment sites, followed by a slow, irreversible incorporation of bound DNA into the cells. Features of the model include a time-dependent exponential decay of functional sites required for DNA uptake and a minimum time for irreversibly bound DNA to enter the cell. We suggest that this model may be useful in studying processes involved in the active transport of DNA across a permeability barrier.     

Phylogenetic utility of ycf1 in orchids: a plastid gene more variable than matK

Plastid DNA sequences have been widely used by systematists for reconstructing plant phylogenies. The utility of any DNA region for phylogenetic analysis is determined by ease of amplification and sequencing, confidence of assessment in phylogenetic character alignment, and by variability across broad taxon sampling. Often, a compromise must be made between using relatively highly conserved coding regions or highly variable introns and intergenic spacers. Analyses of a combination of these types of DNA regions yield phylogenetic structure at various levels of a tree (i.e., along the spine and at the tips of the branches). Here, we demonstrate the phylogenetic utility of a heretofore unused portion of a plastid protein-coding gene, hypothetical chloroplast open reading frame 1 (ycf1), in orchids. All portions of ycf1 examined are highly variable, yet alignable across Orchidaceae, and are phylogenetically informative at the level of species. In Orchidaceae, ycf1 is more variable than matK both in total number of parsimony informative characters and in percent variability. The nrITS region is more variable than ycf1, but is more difficult to align. Although we only demonstrate the phylogenetic utility of ycf1 in orchids, it is likely to be similarly useful among other plant taxa.     

Drought impacts and recovery are driven by local variation in species turnover

In forest systems, drought acts as a disturbance that can generate impacts on community structure and composition at multiple scales. This study focused on a 2-month drought event within an early successional forest system to determine the mechanism(s) of community response to, and recovery from, drought. Drought induced a 28% decline in neighborhood species richness and cover as a result of decreased colonization and increased extinction rates. Following drought, neighborhood richness quickly recovered via increased colonization rates while extinction rates were unaltered. Drought had little long-term effect on neighborhood structure (species richness and cover) and generated only subtle changes in neighborhood composition. Ruderal (annual and biennial) species were more likely to change (increase or decrease) in cover and frequency than the more stress tolerant perennial and woody species. However, population dynamics appeared to be generally driven by stochastic species turnover among fields and not by uniform shifts in species performance across the site. Although drought impacts and recovery appeared predictable at the neighborhood scale, population responses to drought within the site were rather unpredictable. Our findings suggest that stochastic fine-scale processes can generate predictable course-scale dynamics within a disturbed system. The scale-specific mechanisms of community change presented here should be explored in other systems to determine the extent of their generality in driving disturbance impacts on communities.     

Impact of carbon sources on growth and oxalate synthesis by the phytopathogenic fungus <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The impact of various supplemental carbon sources (oxalate, glyoxylate, glycolate, pyruvate, formate, malate, acetate, and succinate) on growth and oxalate formation (i.e., oxalogenesis) by Sclerotinia sclerotiorum was studied. With isolates D-E7, 105, W-B10, and Arg-L of S. sclerotiorum, growth in an undefined broth medium (0.1% soytone; pH 5) with 25 mM glucose and 25 mM supplemental carbon source was increased by the addition of malate and succinate. Oxalate accumulation occurred in the presence of glucose and a supplemental carbon source, with malate, acetate, and succinate supporting the most oxalate synthesis. With S. sclerotiorum Arg-L, oxalate-to-biomass ratios, an indicator of oxalogenic potential, were dissimilar when the organism was grown in the presence of different carbon sources. The highest oxalate-to-biomass ratios were observed with pyruvate, formate, malate, acetate, and succinate. Time-course studies with acetate-supplemented cultures revealed that acetate and glucose consumption by S. sclerotiorum D-E7 coincided with oxalogenesis and culture acidification. By day 5 of incubation, oxalogenesis was halted when cultures reached a pH of 3 and were devoid of acetate. In succinate-supplemented cultures, oxalogenesis essentially paralleled glucose and succinate utilization over the 9-day incubation period; during this time period, culture pH declined but never fell below 4. Overall, these results indicate that carbon sources can regulate the accumulation of oxalate, a key pathogenicity determinant for S. sclerotiorum.     

A core oligosaccharide component from the lipopolysaccharide of Rhizobium trifolii ANU843

The major oligosaccharide from the core region of the lipopolysaccharide from R. trifolii ANU843 was isolated and its structure determined. It is a trisaccharide consisting of two galacturonic acid residues and one 3-deoxy-D-manno-2-octulosonic acid (KDO) residue. The two galacturonic acid residues are terminally linked alpha to the C-4 and C-7 atoms of KDO. This structure was determined through use of 1H- and 13C-n.m.r. spectroscopy, f.a.b.-m.s., and g.l.c.-m.s. techniques. This oligosaccharide had not previously been reported to be present in the lipopolysaccharides from Gram-negative bacteria.     

Metabolic restructuring during energy-limited states: insights from Artemia franciscana embryos and other animals

Many life history stages of animals that experience environmental insults enter developmental arrested states that are characterized by reduced cellular proliferation, with or without a concurrent reduction in overall metabolism. In the case of the most profound metabolic arrest reported in invertebrates, i.e., anaerobic quiescence in Artemia franciscana embryos, acidification of the intracellular milieu is a major factor governing catabolic and anabolic downregulation. Release of ions from intracellular compartments is the source for approximately 50% of the proton equivalents needed for the 1.5 unit acidification that is observed. Recovery from the metabolic arrest requires re-sequestration of the protons with a vacuolar-type ATPase (V-ATPase). The remarkable facet of this mechanism is the ability of embryonic cells to survive the dissipation of intracellular ion gradients. Across many diapause-like states, the metabolic reduction and subsequent matching of energy demand is accomplished by shifting energy metabolism from oxidative phosphorylation to aerobic glycolysis. Molecular pathways that are activated to induce these resilient hypometabolic states include stimulation of the AMP-activated protein kinase (AMPK) and insulin signaling via suite of daf (dauer formation) genes for diapause-like states in nematodes and insects. Contributing factors for other metabolically depressed states involve hypoxia-inducible factor-1 and downregulation of the pyruvate dehydrogenase complex. Metabolic similarities between natural states of stasis and some cancer phenotypes are noteworthy. Reduction of flux through oxidative phosphorylation helps prevent cell death in certain cancer types, similar to the way it increases viability of dauer stages in Caenorhabditis elegans. Mechanisms that underlie natural stasis are being used to pre-condition mammalian cells prior to cell biostabilization and storage.     

Phylogenetic relationships and evolution of Crassulaceae inferred from matK sequence data

Chloroplast gene matK sequence data were used to estimate the phylogeny of 112 species of Crassulaceae sampled from 33 genera and all six recognized subfamilies. Our analyses suggest that five of six subfamilies recognized in the most recent comprehensive classification of the family are not monophyletic. Instead, we recovered a basal split in Crassulaceae between the southern African CRASSULA: clade (Crassuloideae) and the rest of the family (Sedoideae). These results are compatible with recent studies of cpDNA restriction site analyses. Within Sedoideae, four subclades were also recovered: KALANCHOE:, Leucosedum, Acre, and AEONIUM:; evidence also exists for a TELEPHIUM: clade and SEMPERVIVUM: clade. The genus SEDUM: is highly polyphyletic with representatives spread throughout the large Sedoideae clade. Sympetaly and polymerous flowers have arisen multiple times in Crassulaceae and thus are not appropriate characters upon which to base subfamilial limits, as has been done in the past. One floral character, haplostemy, appears to be confined to the well-supported CRASSULA: clade. Our analyses suggest a southern African origin of the family, with subsequent dispersal northward into the Mediterranean region. From there, the family spread to Asia/eastern Europe and northern Europe; two separate lineages of European Crassulaceae subsequently dispersed to North America and underwent substantial diversification. Our analyses also suggest that the original base chromosome number in Crassulaceae is x = 8 and that polyploidy has played an important role in seven clades. Three of these clades are exclusively polyploid (SEMPERVIVUM: clade and two subclades within the KALANCHOE: and AEONIUM: clades), whereas four (Crassula, Telephium, Leucosedum, and ACRE: clades) comprise both diploid and polyploid taxa. Polyploidy is particularly rampant and cytological evolution especially complex in the ACRE: clade.