Elevated CO22 and Leaf Longevity in the C44 Grassland-Dominant Andropogon gerardii

In central U.S. grasslands, plant and ecosystem responses to elevated CO2 are most pronounced when water availability is limited. In a northeast Kansas grassland, responses to elevated CO2 in leaf area, number, development, and longevity were quantified for the tallgrass prairie dominant, Andropogon gerardii. Plants were grown in open-top chambers (OTCs) modified to limit water availability and to maximize responses to elevated CO2. In OTCs with elevated (x2 ambient) levels of CO2, aboveground biomass production and leaf water potentials were increased significantly compared with those of plants in OTCs with ambient CO2. There were no differences in leaf area or leaf number per tiller in A. gerardii in elevated compared with ambient OTCs. However, leaf area in adjacent unchambered plots with greater water availability was significantly higher than in the OTCs. The time required for developing leaves to achieve maximum leaf area was reduced by 29%, and the period of time until leaves senesced was increased by 20% for plants exposed to elevated compared with ambient CO2. Thus, leaves of this C4 grass species expanded more rapidly (6 d) and remained green longer (9 d) when exposed to elevated CO2. Such CO2-mediated increases in leaf longevity in the dominant species may allow this grassland to respond more opportunistically to temporally variable rainfall patterns in high-CO2 environments. These responses should be included in leaf-based simulation models that attempt to mechanistically link physiological alterations to predicted canopy responses to increased CO2.     

Programmed cell death without DNA fragmentation in the jimpy mouse: secreted factors can enhance survival

Jimpy is one of many related mutations affecting the myelin proteolipid protein gene that causes severe hypomyelination in the central nervous system (CNS). Underlying the hypomyelination is a failure of oligodendrocytes (OLs) to differentiate, and the premature death of large numbers of OLs during the developmental period. Previous light and electron microscopic evidence suggested that jimpy OLs die in a manner consistent with programmed cell death. We have used TUNEL staining as a biochemical marker for apoptosis in conjunction with immunostaining for OL and myelin markers. At 13 - 14 days postnatal, a time when the number of dying OLs in jimpy CNS is increased more than five times normal, there are only modest increases (70% in spinal cord; 20% in cerebral cortex) in TUNEL labeled cells in mutant CNS tissues. The results in vitro are similar, and only a small per cent of TUNEL labeled cells have the antigenic phenotype of OLs. The discrepancy between numbers of dying and TUNEL labeled cells suggests either that most jimpy OLs do not undergo programmed cell death or that the biochemical pathways leading to their death do not involve DNA fragmentation which is detected by the TUNEL method. We also present evidence that jimpy OLs show increased survival and enhanced differentiation when they are grown in vitro in medium conditioned by cells lines which express products of the proteolipid protein gene. Cell lines expressing proteolipid protein and the alternatively spliced DM20 protein have differential effects on cell numbers and production of myelin-like membranes.     

TGF-beta1 regulation of dendritic cells

Dendritic cells (DCs) represent antigen-presenting cell (APC) populations in lymphoid and nonlymphoid organs which are considered to play key roles in the initiation of antigen-specific T-cell proliferation. According to current knowledge, the net outcome of T-cell immune responses seems to be significantly influenced by the activation stage of antigen-presenting DCs. Several studies have shown that transforming growth factor-beta 1 (TGF-beta1) inhibits in vitro activation and maturation of DCs. TGF-beta1 inhibits upregulation of critical T-cell costimulatory molecules on the surface of DCs and reduces the antigen-presenting capacity of DCs. Thus, in addition to direct inhibitory effects of TGF-beta1 on effector T lymphocytes, inhibitory effects of TGF-beta1 at the level of APCs may critically contribute to previously characterized immunosuppressive effects of TGF-beta1. In contrast to these negative regulatory effects of TGF-beta1 on function and maturation of lymphoid tissue type DCs, certain subpopulations of immature DCs in nonlymphoid tissues are positively regulated by TGF-beta1 signaling. In particular, epithelial-associated DC populations seem to critically require TGF-beta1 stimulation for development and function. Recent studies established that TGF-beta1 stimulation is absolutely required for the development of epithelial Langerhans cells (LCs) in vitro and in vivo. Furthermore, TGF-beta1 seems to enhance antigen processing and costimulatory functions of epithelial LCs.     

Early development of the motor and premotor circuitry of a sexually dimorphic vocal pathway in a teleost fish

The plainfin midshipman fish (Porichthys notatus) has a caudal hindbrain vocal motor circuit that has been proposed to share a common embryonic origin with the hindbrain vocal networks of other vertebrates. In midshipman, this vocal circuit includes three groups of neurons: sonic motor, pacemaker, and ventral medullary. Here, transneuronal transport of biocytin or neurobiotin was used to delineate the early ontogeny of the three hindbrain vocal nuclei and their pattern of connectivity. The organization of the vocal nuclei was studied in animals beginning soon after hatching until the nuclei have the adult phenotype at the time fish become free-swimming. There is a clear sequence of events whereby motoneurons establish their connections with the sonic muscle prior to establishing connections with premotor neurons; developmental milestones of the vocal pathway parallel those of the sonic muscle. The results also indicate that sexual differentiation of the vocal motor system in midshipman begins early in development, well before any evidence of sexual maturation. Embryonic males and females differ in the relationship between soma size and body length for the three hindbrain nuclei. Males are also more variable than females in body mass, volume of the sonic motor nucleus, and motoneuron cell size.     

Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2+3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (<0.049×10⁻⁷). The frequency of HPRT exons 2+3 deletions was 0.10×10⁻⁷ per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%–70%) and previous results using the T-cell cloning assay (2–3×10⁻⁷ per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7×10⁸ leukocytes analyzed), and which occurred at frequencies from <1×10⁻⁷ (undetected) to 1.6×10⁻⁴ among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2+3 deletions in the leukocytes of the newborns, from 0.07×10⁻⁷ to 0.23×10⁻⁷. We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2+3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.     

A note on power approximations for the transmission/disequilibrium test

The transmission/disequilibrium test (TDT) is a popular method for detection of the genetic basis of a disease. Investigators planning such studies require computation of sample size and power, allowing for a general genetic model. Here, a rigorous method is presented for obtaining the power approximations of the TDT for samples consisting of families with either a single affected child or affected sib pairs. Power calculations based on simulation show that these approximations are quite precise. By this method, it is also shown that a previously published power approximation of the TDT is erroneous.     

The staphylococcal alpha-toxin pore has a flexible conformation

The alpha-toxin from Staphylococcus aureus undergoes several conformational changes from the time it is released from the bacterium to the moment it forms a channel in the plasma membrane of its target cell. It is initially a soluble monomer, which undergoes membrane binding and oligomerization into a heptameric ring and finally inserts into the lipid bilayer to form a pore. Here we have analyzed the stability of different forms of the alpha-toxin (monomer as well as heptamers in solution, bound to the membrane and membrane-inserted) by differential scanning calorimetry and limited proteolysis. Data presented here show that, in contrast to both the membrane-bound prepore complex and the monomer in solution, the membrane-inserted alpha-toxin channel does not undergo cooperative unfolding and is highly susceptible to proteases. These observations suggest that the channel has a looser conformation. Interestingly, resistance to proteases could be recovered upon solubilization of the channel, indicating that the loss of rigid tertiary packing only occurred upon membrane insertion. Far-UV CD data, however, suggest that the transmembrane beta-barrel must be stably folded and that therefore only the Cap and Rim domains of the channel are loosely packed. All together, our data show that the alpha-toxin channel is not a rigid complex within the membrane but adopts a rather flexible conformation.     

QTL mapping of <Emphasis Type="Italic">Sclerotinia</Emphasis> midstalk-rot resistance in sunflower

In many sunflower-growing regions of the world, Sclerotinia sclerotiorum (Lib.) de Bary is the major disease of sunflower (Helianthus annuus L.). In this study, we mapped and characterized quantitative trait loci (QTL) involved in resistance to S. sclerotiorum midstalk rot and two morphological traits. A total of 351 F₃ families developed from a cross between a resistant inbred line from the germplasm pool NDBLOS and the susceptible line CM625 were assayed for their parental F₂ genotype at 117 codominant simple sequence repeat markers. Disease resistance of the F₃ families was screened under artificial infection in field experiments across two sowing times in 1999. For the three resistance traits (leaf lesion, stem lesion, and speed of fungal growth) and the two morphological traits, genotypic variances were highly significant. Heritabilities were moderate to high (h²=0.55–0.89). Genotypic correlations between resistance traits were highly significant (P<0.01) but moderate. QTL were detected for all three resistance traits, but estimated effects at most QTL were small. Simultaneously, they explained between 24.4% and 33.7% of the genotypic variance for resistance against S. sclerotiorum. Five of the 15 genomic regions carrying a QTL for either of the three resistance traits also carried a QTL for one of the two morphological traits. The prospects of marker-assisted selection (MAS) for resistance to S. sclerotiorum are limited due to the complex genetic architecture of the trait. MAS can be superior to classical phenotypic selection only with low marker costs and fast selection cycles.     

Development of alternate ssu-rRNA probing strategies for characterizing aquatic microbial communities

Plastids in phytoplankton retain prokaryote-like DNA sequences that may generate false-positive signals from eubacterial small subunit (ssu) rRNA oligonucleotide probes, resulting in the overestimation of bacterial activity in aquatic microbial communities. To assess the extent of possible plastid-associated binding to eubacterial signals, we performed an extensive database search, flask experiments using algal and cyanobacterial pure cultures, and field trials on five common eubacterial probes: S-D-Bact-008-a-A-19, S-D-Bact-338-a-A-18, S-D-Bact-785-a-A-19, S-D-Bact-927-a-A-17, and S-D-Bact-1088-a-A-20. The database search and laboratory tests showed significant potential for binding among most bacterial probes and organelle ssu-rRNA. However, we propose two probing strategies to overcome this problem. First, one could use Bact-785 and Bact-338 in tandem, with the plastid component being estimated as the difference between the two signals (Bact-338 has approximately 70% overlap with known plastid sequences). Alternately, one might use Bact-338 as the primary eubacterial probe, but then use Cyan-785-a-A-19 (a probe that binds significantly to plastid rRNA) to correct for the plastid-associated false-positive signal. Both strategies would use a eukaryotic probe (S-D-Euca-1379-a-A-16) and Cyan-785-b-A-19 (a probe for most cyanobacteria) to further segregate rRNA signals. Trials were successfully performed using the strategies on samples from a recent field study.     

Comparison of methods to detect biosurfactant production by diverse microorganisms

Three methods to detect biosurfactant production, drop collapse, oil spreading, and blood agar lysis, were compared for their ease of use and reliability in relation to the ability of the cultures to reduce surface tension. The three methods were used to test for biosurfactant production in 205 environmental strains with different phylogenetic affiliations. Surface tension of select strains that gave conflicting results with the above three methods was also measured. Sixteen percent of the strains that lysed blood agar tested negative for biosurfactant production with the other two methods and had little reduction in surface tension (values above 60 mN/m). Thirty eight percent of the strains that did not lyse blood agar tested positive for biosurfactant production with the other two methods and had surface tension values as low as 35 mN/m. There was a very strong, negative, linear correlation between the diameter of clear zone obtained with the oil spreading technique and surface tension (rs = -0.959) and a weaker negative correlation between drop collapse method and surface tension (rs = -0.82), suggesting that the oil spreading technique better predicted biosurfactant production than the drop collapse method. The use of the drop collapse method as a primary method to detect biosurfactant producers, followed by the determination of the biosurfactant concentration using the oil spreading technique, constitutes a quick and easy protocol to screen and quantify biosurfactant production. The large number of false negatives and positives obtained with the blood agar lysis method and its poor correlation to surface tension (rs = -0.15) demonstrated that it is not a reliable method to detect biosurfactant production.